Plants, seeds and oils having an elevated total monounsaturated fatty acid content

ABSTRACT

Plants, seeds and oils having a total long chain monounsaturated content of at least about 82% and an erucic acid content of at least about 15% are described. Methods for producing plants having the profiled fatty acid content are also described.

TECHNICAL FIELD

[0001] This invention relates to fatty acid desaturases and nucleicacids encoding desaturase proteins. More particularly, the inventionrelates to nucleic acids encoding delta-12 and delta-15 fatty aciddesaturase proteins that affect fatty acid composition in plants,polypeptides produced from such nucleic acids and plants expressing suchnucleic acids.

BACKGROUND OF THE INVENTION

[0002] Many breeding studies have been conducted to improve the fattyacid profile of Brassica varieties. Pleines and Freidt, Fat Sci.Technol., 90(5), 167-171 (1988) report plant lines with reduced C_(18:3)levels (2.5-5.8%) combined with high oleic content (73-79%). Rakow andMcGregor, J. Amer. Oil Chem. Soc., 50, 400-403 (October 1973) discussproblems associated with selecting mutants for linoleic and linolenicacids. In Can. J. Plant Sci., 68, 509-511 (April 1988) Stellar summerrape producing seed oil with 3% linolenic acid and 28% linoleic acid isdisclosed. Roy and Tarr, Z. Pflanzenzuchtg, 95(3), 201-209 (1985) reporttransfer of genes through an interspecific cross from Brassica junceainto Brassica napus resulting in a reconstituted line combining highlinoleic with low linolenic acid content. Roy and Tarr, Plant Breeding,98, 89-96 (1987) discuss prospects for development of B. napus L. havingimproved linolenic and linolenic acid content. European Patentapplication 323,753 published Jul. 12, 1989 discloses seeds and oilshaving greater than 79% oleic acid combined with less than 3.5%linolenic acid. Canvin, Can. J. Botany, 43, 63-69 (1965) discusses theeffect of temperature on the fatty acid composition of oils from severalseed crops including rapeseed.

[0003] Mutations typically are induced with extremely high doses ofradiation and/or chemical mutagens (Gaul, H. Radiation Botany (1964)4:155-232). High dose levels which exceed LD₅₀, and typically reachLD₉₀, led to maximum achievable mutation rates. In mutation breeding ofBrassica varieties, high levels of chemical mutagens alone or combinedwith radiation have induced a limited number of fatty acid mutations(Rakow, G. Z. Pflanzenzuchtg (1973) 69:62-82). The low α-linolenic acidmutation derived from the Rakow mutation breeding program did not havedirect commercial application because of low seed yield. The firstcommercial cultivar using the low α-linolenic acid mutation derived in1973 was released in 1988 as the variety Stellar (Scarth, R. et al.,Can. J. Plant Sci. (1988) 68:509-511). Stellar was 20% lower yieldingthan commercial cultivars at the time of its release.

[0004] Alterations in fatty acid composition of vegetable oils isdesirable for meeting specific food and industrial uses. For example,Brassica canola varieties with increased monounsaturate levels (oleicacid) in the seed oil, and products derived from such oil, would improvelipid nutrition. Canola lines which are low in polyunsaturated fattyacids and high in oleic acid tend to have higher oxidative stability,which is a useful trait for the retail food industry. Useful traits ofvegetable oils for industrial uses like lubrication fluids includedesirable low temperature behavior such as low pour point and low cloudpoint along with very high oxidative stability.

[0005] Delta-12 fatty acid desaturase (also known as oleic desaturase)is involved in the enzymatic conversion of oleic acid to linoleic acid.Delta-15 fatty acid desaturase (also known as linoleic acid desaturase)is involved in the enzymatic conversion of linoleic acid to α-linolenicacid. A microsomal delta-12 desaturase has been cloned and characterizedusing T-DNA tagging. Okuley, et al., Plant Cell 6:147-158 (1994). Thenucleotide sequences of higher plant genes encoding microsomal delta-12fatty acid desaturase are described in Lightner et al., WO94/11516.Sequences of higher plant genes encoding microsomal and plastid delta-15fatty acid desaturases are disclosed in Yadav, N., et al., PlantPhysiol., 103:467-476 (1993), WO 93/11245 and Arondel, V. et al.,Science, 258:1353-1355 (1992).

SUMMARY OF THE INVENTION

[0006] Triacylglycerols containing fatty acids with heterogenous chainlengths and with high monounsaturate levels can provide useful traitsfor industrial purposes. Plants with fatty acid compositions that havehigh monounsaturate levels and heterogenous chain lengths would providea source of industrial oils for uses such as lubrication.

[0007] In one aspect, the invention features a Brassica plant, andprogeny thereof, producing seeds having a long chain monounsaturatedfatty acid content of at least about 82% and an erucic acid content ofat least about 15% based on total fatty acid composition. The oleic acidand eicosenoic acid content of the seeds is at least about 37% and atleast about 14%, based on total fatty acid composition, respectively.The saturated fatty acid content of such seeds is less than 7% and thepolyunsaturated fatty acid content is less than about 11%.

[0008] In some embodiments, the plants have a monounsaturated fatty acidcontent of from about 85% to about 90% and an erucic acid content of atleast about 15% based on total fatty acid composition. In such plants,the oleic acid content can be at least about 42% and in particular, fromabout 47% to about 56% based on total fatty acid composition. The erucicacid content is from about 17% to about 31%, and the eicosenoic acidcontent is from about 15% to about 21%.

[0009] The invention also features a Brassica seed oil having a longchain monounsaturated fatty acid content of at least about 82% and anerucic acid content of at least about 15% based on total fatty acidcomposition. Such oils can have an oleic acid and eicosenoic acidcontent of at least about 14% and 37%, respectively, based on totalfatty acid composition. The saturated fatty acid content is less thanabout 7%. The polyunsaturated fatty acid content is less than about 11%and in particular embodiments, less than 9%, based on total fatty acidcomposition.

[0010] In some embodiments, the Brassica seed oil contains a long chainmonounsaturated fatty acid content of from about 85% to about 90%. Insuch oils, the oleic acid content is at least about 42%, and inparticular embodiments, is from about 47% to about 56%, based on totalfatty acid composition. The erucic acid and eicosenoic acid content isfrom about 17% to about 31% and from about 15% to about 21%,respectively, based on total fatty acid composition.

[0011] The invention also features a method of producing plants having along chain monounsaturated fatty acid content of at least about 82% andan erucic acid content of at least about 15%, based on total fatty acidcomposition. The methods include crossing a first plant line with asecond plant line and selecting progeny with the desired fatty acidcomposition. The first plant line has an erucic acid content of at leastabout 45%. The second plant line has an oleic acid content of at leastabout 84%.

Brief Description of the Sequence Listing

[0012] SEQ ID NO:1 shows a hypothetical DNA sequence of a Brassica Fad2gene. SEQ ID NO:2 is the deduced amino acid sequence of SEQ ID NO: 1.

[0013] SEQ ID NO:3 shows a hypothetical DNA sequence of a Brassica Fad2gene having a mutation at nucleotide 316. SEQ ID NO:4 is the deducedamino acid sequence of SEQ ID NO:3.

[0014] SEQ ID NO:5 shows a hypothetical DNA sequence of a Brassica Fad2gene. SEQ ID NO:6 is the deduced amino acid sequence of SEQ ID NO:5.

[0015] SEQ ID NO:7 shows a hypothetical DNA sequence of a Brassica Fad2gene having a mutation at nucleotide 515. SEQ ID NO:8 is the deducedamino acid sequence of SEQ ID NO:7.

[0016] SEQ ID NO:9 shows the DNA sequence for the coding region of awild type Brassica Fad2-D gene. SEQ ID-NO:10 is the deduced amino acidsequence for SEQ ID NO:9.

[0017] SEQ ID NO:11 shows the DNA sequence for the coding region of theIMC 129 mutant Brassica Fad2-D gene. SEQ ID NO:12 is the deduced aminoacid sequence for SEQ ID NO:11.

[0018] SEQ ID NO:13 shows the DNA sequence for the coding region of awild type Brassica Fad2-F gene. SEQ ID NO:14 is the deduced amino acidsequence for SEQ ID NO:13.

[0019] SEQ ID NO:15 shows the DNA sequence for the coding region of theQ508 mutant Brassica Fad2-F gene. SEQ ID NO:16 is the deduced amino acidsequence for SEQ ID NO:15.

[0020] SEQ ID NO:17 shows the DNA sequence for the coding region of theQ4275 mutant Brassica Fad2-F gene. SEQ ID NO: 18 is the deduced aminoacid sequence for SEQ ID NO:17.

BRIEF DESCRIPTION OF THE FIGURES

[0021]FIG. 1 is a histogram showing the frequency distribution of seedoil oleic acid (C_(18:1)) content in a segregating population of a Q508X Westar cross. The bar labeled WSGA 1A represents the C_(18:1) contentof the Westar parent. The bar labeled Q508 represents the C_(18:1)content of the Q508 parent.

[0022]FIG. 2 shows the nucleotide sequences for a Brassica Fad2-D wildtype gene (Fad2-D wt), IMC129 mutant gene (Fad2-D GA316 IMC129), Fad2-Fwild type gene (Fad2-F wt), Q508 mutant gene (Fad2-F TA515 Q508) andQ4275 mutant gene (Fad2-F GA908 Q4275).

[0023]FIG. 3 shows the deduced amino acid sequences for thepolynucleotides of FIG. 2.

[0024]FIG. 4 is a schematic of a breeding procedure used to produceBrassica plants having a high erucic acid and a high oleic acid content.

DETAILED DESCRIPTION

[0025] All percent fatty acids herein are percent by weight of the oilof which the fatty acid is a component.

[0026] As used herein, a “line” is a group of plants that display littleor no genetic variation between individuals for at least one trait. Suchlines may be created by several generations of self-pollination andselection, or vegetative propagation from a single parent using tissueor cell culture techniques. As used herein, the term “variety” refers toa line which is used for commercial production.

[0027] The term “mutagenesis” refers to the use of a mutagenic agent toinduce random genetic mutations within a population of individuals. Thetreated population, or a subsequent generation of that population, isthen screened for usable trait(s) that result from the mutations. A“population” is any group of individuals that share a common gene pool.As used herein “M₀” is untreated seed. As used herein, “M₁” is the seed(and resulting plants) exposed to a mutagenic agent, while “M₂” is theprogeny (seeds and plants) of self-pollinated M₁ plants, “M₃” is theprogeny of self-pollinated M₂ plants, and “M₄” is the progeny ofself-pollinated M₃ plants. “M₅” is the progeny of self-pollinated M₄plants. “M₆”, “M₇”, etc. are each the progeny of self-pollinated plantsof the previous generation. The term “selfed” as used herein meansself-pollinated.

[0028] “Stability” or “stable” as used herein means that with respect toa given fatty acid component, the component is maintained fromgeneration to generation for at least two generations and preferably atleast three generations at substantially the same level, e.g.,preferably ±5%. The method of invention is capable of creating lineswith improved fatty acid compositions stable up to ±5% from generationto generation. The above stability may be affected by temperature,location, stress and time of planting. Thus, comparison of fatty acidprofiles should be made from seeds produced under similar growingconditions. Stability may be measured based on knowledge of priorgeneration.

[0029] Intensive breeding has produced certain Brassica plants whoseseed oil contains less than 2% erucic acid. The same varieties have alsobeen bred so that the defatted meal contains less than 30 μmolglucosinolates/gram. “Canola” as used herein refers to plant seeds oroils which contain less than 2% erucic acid (C_(22:1)), and result in adefatted meal with less than 30 μmol glucosinolates/gram.

[0030] Applicants have discovered plants with mutations in a delta-12fatty acid desaturase gene. Such plants have useful alterations in thefatty acid compositions of the seed oil. Such mutations confer, forexample, an elevated oleic acid content, a decreased, stabilizedlinoleic acid content, or both elevated oleic acid and decreased,stabilized linoleic acid content.

[0031] Applicants have further discovered plants with mutations in adelta-15 fatty acid desaturase gene. Such plants have useful alterationsin the fatty acid composition of the seed oil, e.g., a decreased,stabilized level of α-linolenic acid.

[0032] Applicants have further discovered isolated nucleic acidfragments (polynucleotides) comprising sequences that carry mutationswithin the coding sequence of delta-12 or delta-15 fatty aciddesaturases. The mutations confer desirable alterations in fatty acidlevels in the seed oil of plants carrying such mutations. Delta-12 fattyacid desaturase is also known as omega-6 fatty acid desaturase and issometimes referred to herein as Fad2 or 12-DES. Delta-15 fatty aciddesaturase is also known on omega-3 fatty acid desaturase and issometimes referred to herein as Fad3 or 15-DES.

[0033] A nucleic acid fragment of the invention may be in the form ofRNA or in the form of DNA, including cDNA, synthetic DNA or genomic DNA.The DNA may be double-stranded or single-stranded, and ifsingle-stranded, can be either the coding strand or non-coding strand.An RNA analog may be, for example, mRNA or a combination of ribo- anddeoxyribonucleotides. Illustrative examples of a nucleic acid fragmentof the invention are the mutant sequences shown in FIG. 3.

[0034] A nucleic acid fragment of the invention contains a mutation in amicrosomal delta-12 fatty acid desaturase coding sequence or a mutationin a microsomal delta-15 fatty acid desaturase coding sequence. Such amutation renders the resulting desaturase gene product non-functional inplants, relative to the function of the gene product encoded by thewild-type sequence. The non-functionality of the delta-12 desaturasegene product can be inferred from the decreased level of reactionproduct (linoleic acid) and increased level of substrate (oleic acid) inplant tissues expressing the mutant sequence, compared to thecorresponding levels in plant tissues expressing the wild-type sequence.The non-functionality of the delta-15 desaturase gene product can beinferred from the decreased level of reaction product (α-linolenic acid)and the increased level of substrate (linoleic acid) in plant tissuesexpressing the mutant sequence, compared to the corresponding levels inplant tissues expressing the wild-type sequence.

[0035] A nucleic acid fragment of the invention may comprise a portionof the coding sequence, e.g., at least about 10 nucleotides, providedthat the fragment contains at least one mutation in the coding sequence.The length of a desired fragment depends upon the purpose for which thefragment will be used, e.g., PCR primer, site-directed mutagenesis andthe like. In one embodiment, a nucleic acid fragment of the inventioncomprises the full length coding sequence of a mutant delta-12 or mutantdelta-15 fatty acid desaturase, e.g., the mutant sequences of FIG. 3. Inother embodiments, a nucleic acid fragment is about 20 to about 50nucleotides (or base pairs, bp), or about 50 to about 500 nucleotides,or about 500 to about 1200 nucleotides in length.

[0036] Desirable alterations in fatty acid levels in the seed oil ofplants can be produced using a ribozyme. Ribozyme molecules designed tocleave delta-12 or delta-15 desaturase mRNA transcripts can be used toprevent expression of delta-12 or delta-15 desaturases. While variousribozymes that cleave mRNA at site-specific recognition sequences can beused to destroy desaturase mRNAs, hammerhead ribozymes are particularlyuseful. Hammerhead ribozymes cleave mRNAs at locations dictated byflanking regions that form complementary base pairs with the targetmRNA. The sole requirement is that the target RNA contain a 5′-UG-3′nucleotide sequence. The construction and production of hammerheadribozymes is well known in the art. See, for example, U.S. Pat. No.5,254,678. Hammerhead ribozyme sequences can be embedded in a stable RNAsuch as a transfer RNA (tRNA) to increase cleavage efficiency in vivo.Perriman, R. et al., Proc. Natl. Acad. Sci. USA, 92(13):6175-6179(1995); de Feyter, R. and Gaudron, J., Methods in Molecular Biology,Vol. 74, Chapter 43, “Expressing Ribozymes in Plants”, Edited by Turner,P. C, Humana Press Inc., Totowa, N.J. RNA endoribonucleases such as theone that occurs naturally in Tetrahymena thermophila, and which havebeen described extensively by Cech and collaborators are also useful.See, for example, U.S. Pat. No. 4,987,071.

[0037] A mutation in a nucleic acid fragment of the invention may be inany portion of the coding sequence that renders the resulting geneproduct non-functional. Suitable types of mutations include, withoutlimitation, insertions of nucleotides, deletions of nucleotides, ortransitions and transversions in the wild-type coding sequence. Suchmutations result in insertions of one or more amino acids, deletions ofone or more amino acids, and non-conservative amino acid substitutionsin the corresponding gene product. In some embodiments, the sequence ofa nucleic acid fragment may comprise more than one mutation or more thanone type of mutation.

[0038] Insertion or deletion of amino acids in a coding sequence may,for example, disrupt the conformation of essential alpha-helical orbeta-pleated sheet regions of the resulting gene product. Amino acidinsertions or deletions may also disrupt binding or catalytic sitesimportant for gene product activity. It is known in the art that theinsertion or deletion of a larger number of contiguous amino acids ismore likely to render the gene product non-functional, compared to asmaller number of inserted or deleted amino acids.

[0039] Non-conservative amino acid substitutions may replace an aminoacid of one class with an amino acid of a different class.Non-conservative substitutions may make a substantial change in thecharge or hydrophobicity of the gene product. Non-conservative aminoacid substitutions may also make a substantial change in the bulk of theresidue side chain, e.g., substituting an alanyl residue for a isoleucylresidue.

[0040] Examples of non-conservative substitutions include thesubstitution of a basic amino acid for a non-polar amino acid, or apolar amino acid for an acidic amino acid. Because there are only 20amino acids encoded in a gene, substitutions that result in anon-functional gene product may be determined by routineexperimentation, incorporating amino acids of a different class in theregion of the gene product targeted for mutation.

[0041] Preferred mutations are in a region of the nucleic acid encodingan amino acid sequence motif that is conserved among delta-12 fatty aciddesaturases or delta-15 fatty acid desaturases, such as aHis-Xaa-Xaa-Xaa-His motif (Tables 1-3). An example of a suitable regionhas a conserved HECGH motif that is found, for example, in nucleotidescorresponding to amino acids 105 to 109 of the Arabidopsis and Brassicadelta-12 desaturase sequences, in nucleotides corresponding to aminoacids 101 to 105 of the soybean delta-12 desaturase sequence and innucleotides corresponding to amino acids 111 to 115 of the maizedelta-12 desaturase sequence. See e.g., WO 94/115116; Okuley et al.,Plant Cell 6:147-158 (1994). The one letter amino acid designations usedherein are described in Alberts, B. et al., Molecular Biology of theCell, 3rd edition, Garland Publishing, New York, 1994. Amino acidsflanking this motif are also highly conserved among delta-12 anddelta-15 desaturases and are also suitable candidates for mutations infragments of the invention.

[0042] An illustrative embodiment of a mutation in a nucleic acidfragment of the invention is a Glu to Lys substitution in the HECGHmotif of a Brassica microsomal delta-12 desaturase sequence, either theD form or the F form. This mutation results in the sequence HECGH beingchanged to HKCGH as seen by comparing SEQ ID NO:10 (wild-type D form) toSEQ ID NO: 12 (mutant D form). A similar mutation in other Fad-2sequences is contemplated to result in a non-functional gene product.(Compare SEQ ID NO:2 to SEQ ID NO:4).

[0043] A similar motif may be found at amino acids 101 to 105 of theArabidopsis microsomal delta-15 fatty acid desaturase, as well as in thecorresponding rape and soybean desaturases (Table 5). See, e.g., WO93/11245; Arondel, V. et al., Science, 258:1153-1155 (1992); Yadav, N.et al., Plant Physiol., 103:467-476 (1993). Plastid delta-15 fatty acidshave a similar motif (Table 5).

[0044] Among the types of mutations in an HECGH motif that render theresulting gene product non-functional are non-conservativesubstitutions. An illustrative example of a non-conservativesubstitution is substitution of a glycine residue for either the firstor second histidine. Such a substitution replaces a charged residue(histidine) with a non-polar residue (glycine). Another type of mutationthat renders the resulting gene product non-functional is an insertionmutation, e.g., insertion of a glycine between the cysteine and glutamicacid residues in the HECGH motif.

[0045] Other regions having suitable conserved amino acid motifs includethe HRRHH motif shown in Table 2, the HRTHH motif shown in Table 6 andthe HVAHH motif shown in Table 3. See, e.g., WO 94/115116; Hitz, W. etal., Plant Physiol., 105:635-641 (1994); Okuley, J., et al., supra; andYadav, N. et al., supra An illustrative example of a mutation in theregion shown in Table 3 is a mutation at nucleotides corresponding tothe codon for glycine (amino acid 303 of B. napus). A non-conservativeGly to Glu substitution results in the amino acid sequence DRDYGILNKVbeing changed to sequence DRDYEILNKV (compare wild-type F form SEQ IDNO:14 to mutant Q4275 SEQ ID NO:18, FIG. 3).

[0046] Another region suitable for a mutation in a delta-12 desaturasesequence contains the motif KYLNNP at nucleotides corresponding to aminoacids 171 to 175 of the Brassica desaturase sequence. An illustrativeexample of a mutation is this region is a Leu to His substitution,resulting in the amino acid sequence (Table 4) KYHNN (compare wild-typeFad2-F SEQ ID NO:14 to mutant SEQ ID NO:16). A similar mutation in otherFad-2 amino acid sequences is contemplated to result in a non-functionalgene product. (Compare SEQ ID NO:6 to SEQ ID NO:8). TABLE 1 Alignment ofAmino Acid Sequences from Microsomal Delta-12 Fatty Acid DesaturasesPosi- Species tion Amino Acid Sequence Arabidopsis 100- IWVIAHECGHHAFSDYQWLD DTVGLIFHSF thaliana 129 Glycine max  96- VWVIAHECGHHAFSKYQWVD DVVGLTLHST 125 Zea mays 106- VWVIAHECGH RAFSDYSLLD DVVGLVLHSS135 Ricinus   1- WVMAHDCGH HAFSDYQLLD DVVGLILHSC communis ^(a)  29Brassica 100- VWVIAHECGH HAFSDYQWLD DTVGLIFHS napus D 128 Brassica 100-VWVIAHECGH HAFSDYQWLD DTVGLIFHS napus F 128

[0047] TABLE 2 Alignment of Amino Acid Sequences from MicrosomalDelta-12 Fatty Acid Desaturases Species Position Amino Acid SeauenceArabidopsis 130-158 LLVPYFSWKY SHRRHHSNTG SLERDEVFV thaliana Glycine max126-154 LLVPYFSWKI SHRRHHSNTG SLDRDEVFV Zea mays 136-164 LMVPYFSWKYSHRRHHSNTG SLERDEVFV Ricinus 30-58 LLVPYFSWKH SHRRHHSNTG SLERDEVFVcommunis ^(a) Brassica 130-158 LLVPYFSWKY SHRRHHSNTG SLERDEVFV napus DBrassica 130-158 LLVPYFSWKY SHRRHHSNTG SLERDEVFV napus F

[0048] TABLE 3 Alignment of Amino Acid Sequences from MicrosomalDelta-12 Fatty Acid Desaturases Species Position Amino Acid SequenceArabidopsis thaliana 298-333 DRDYGILNKV FHNITDTHVA HHLFSTMPHY NAMEATGlycine max 294-329 DRDYGILNKV FHNITDTHVA HHLFSTMPHY HAMEAT Zea mays305-340 DRDYGILNRV FHNITDTHVA HHLFSTMPHY HAMEAT Ricinus communis ^(a)198-224 DRDYGILNKV FHNITDTQVA HHLF TMP Brassica napus D 299-334DRDYGILNKV FHNITDTHVA HHLFSTMPHY HAMEAT Brassica napus F 299-334DRDYGILNXV FHNITDTHVA HHLFSTMPHY HAMEAT

[0049] TABLE 4 Alignment of Conserved Amino Acids from MicrosomalDelta-12 Fatty Acid Desaturases Species Position Amino Acid SequenceArabidopsis thaliana 165-180 IKWYGKYLNN PLGRIM Glycine max 161-176VAWFSLYLNN PLGRAV Zea mays 172-187 PWYTPYVYNN PVGRVV Ricinus communis^(a)  65-80 IRWYSKYLNN PPGRIM Brassica napus D 165-180 IKWYGKYLNN PLGRTVBrassica napus F 165-180 IKWYGKYLNN PLGRTV

[0050] TABLE 5 Alignment of Conserved Amino Acids from Plastid andMicrosomal Delta-15 Fatty Acid Desaturases Species Position Amino AcidSequence Arabidopsis 156-177 WALFVLGHD CGHGSFSNDP KLN thaliana ^(a)Brassica 114-135 WALFVLGHD CGHGSFSNDP RLN napus ^(a) Glycine max ^(a)164-185 WALFVLGHD CGHGSFSNNS KLN Arabidopsis  94-115 WAIFVLGHDCGHGSFSDIP LLN thaliana Brassica  87-109 WALFVLGHD CGHGSFSNDP RLN napusGlycine max  93-114 WALFVLGHD CGHGSFSDSP PLN

[0051] TABLE 6 Alignment of Conserved Amino Acids from Plastid andMicrosomal Delta-15 Fatty Acid Desaturases Posi- Species tion Amino AcidSequence A. thaliana ^(a) 188- ILVPYHGWRI SHRTHHQNHG HVENDESWH 216 B.napus ^(a) 146- ILVPYHGWRI SHRTHHQNHG HVENDESWH 174 Glycine max ^(a)196- ILVPYHGWRI SHRTHHQNHG HAENDESWH 224 A. thaliana 126- ILVPYHGWRISHRTHHQNHG RVENDESWV 154 Brassica 117- ILVPYHGWRI SHRTHHQNHG HVENDESWVnapus 145 Glycine max 125- ILVPYHGWRI SHRTHHQNHG HIENDESWV 153

[0052] The conservation of amino acid motifs and their relativepositions indicates that regions of a delta-12 or delta-15 fatty aciddesaturase that can be mutated in one species to generate anon-functional desaturase can be mutated in the corresponding regionfrom other species to generate a non-functional delta-12 desaturase ordelta-15 desaturase gene product in that species.

[0053] Mutations in any of the regions of Tables 1-6 are specificallyincluded within the scope of the invention and are substantiallyidentical to those mutations exemplified herein, provided that suchmutation (or mutations) renders the resulting desaturase gene productnon-functional, as discussed hereinabove.

[0054] A nucleic acid fragment containing a mutant sequence can begenerated by techniques known to the skilled artisan. Such techniquesinclude, without limitation, site-directed mutagenesis of wild-typesequences and direct synthesis using automated DNA synthesizers.

[0055] A nucleic acid fragment containing a mutant sequence can also begenerated by mutagenesis of plant seeds or regenerable plant tissue by,e.g., ethyl methane sulfonate, X-rays or other mutagens. Withmutagenesis, mutant plants having the desired fatty acid phenotype inseeds are identified by known techniques and a nucleic acid fragmentcontaining the desired mutation is isolated from genomic DNA or RNA ofthe mutant line. The site of the specific mutation is then determined bysequencing the coding region of the delta-12 desaturase or delta-15desaturase gene. Alternatively, labeled nucleic acid probes that arespecific for desired mutational events can be used to rapidly screen amutagenized population.

[0056] The disclosed method may be applied to all oilseed Brassicaspecies, and to both Spring and Winter maturing types within eachspecies. Physical mutagens, including but not limited to X-rays, UVrays, and other physical treatments which cause chromosome damage, andother chemical mutagens, including but not limited to ethidium bromide,nitrosoguanidine, diepoxybutane etc. may also be used to inducemutations. The mutagenesis treatment may also be applied to other stagesof plant development, including but not limited to cell cultures,embryos, microspores and shoot apices.

[0057] “Stable mutations” as used herein are defined as M₅ or moreadvanced lines which maintain a selected altered fatty acid profile fora minimum of three generations, including a minimum of two generationsunder field conditions, and exceeding established statistical thresholdsfor a minimum of two generations, as determined by gas chromatographicanalysis of a minimum of 10 randomly selected seeds bulked together.Alternatively, stability may be measured in the same way by comparing tosubsequent generations. In subsequent generations, stability is definedas having similar fatty acid profiles in the seed as that of the prioror subsequent generation when grown under substantially similarconditions.

[0058] Mutation breeding has traditionally produced plants carrying, inaddition to the trait of interest, multiple, deleterious traits, e.g.,reduced plant vigor and reduced fertility. Such traits may indirectlyaffect fatty acid composition, producing an unstable mutation; and/orreduce yield, thereby reducing the commercial utility of the invention.To eliminate the occurrence of deleterious mutations and reduce the loadof mutations carried by the plant, a low mutagen dose is used in theseed treatments to create an LD30 population. This allows for the rapidselection of single gene mutations for fatty acid traits in agronomicbackgrounds which produce acceptable yields.

[0059] The seeds of several different fatty acid lines have beendeposited with the American Type Culture Collection and have thefollowing accession numbers. Line Accession No. Deposit Date A129.540811 May 25, 1990 A133.1 40812 May 25, 1990 M3032.1 75021 Jun. 7, 1991M3062.8 75025 Jun. 7, 1991 M3028.10 75026 Jun. 7, 1991 IMC130 75446 Apr.16, 1993 Q4275 97569 May 10, 1996 V800655.334 V800655.126 V800654.9

[0060] In some plant species or varieties more than one form ofendogenous microsomal delta-12 desaturase may be found. Inamphidiploids, each form may be derived from one of the parent genomesmaking up the species under consideration. Plants with mutations in bothforms have a fatty acid profile that differs from plants with a mutationin only one form. An example of such a plant is Brassica napus lineQ508, a doubly-mutagenized line containing a mutant D-form of delta-12desaturase (SEQ ID NO:11) and a mutant F-form of delta-12 desaturase(SEQ ID NO:15). Another example is line Q4275, which contains a mutantD-form of delta-12 desaturase (SEQ ID NO:11) and a mutant F-form ofdelta-12 desaturase (SEQ ID NO:17). See FIGS. 2-3.

[0061] Preferred host or recipient organisms for introduction of anucleic acid fragment of the invention are the oil-producing species,such as soybean (Glycine max), rapeseed (e.g., Brassica napus, B. rapaand B. juncea), sunflower (Helianthus annus), castor bean (Ricinuscommunis), corn (Zea mays), and safflower (Carthamus tinctorius).

[0062] A nucleic acid fragment of the invention may further compriseadditional nucleic acids. For example, a nucleic acid encoding asecretory or leader amino acid sequence can be linked to a mutantdesaturase nucleic acid fragment such that the secretory or leadersequence is fused in-frame to the amino terminal end of a mutantdelta-12 or delta-15 desaturase polypeptide. Other nucleic acidfragments are known in the art that encode amino acid sequences usefulfor fusing in-frame to the mutant desaturase polypeptides disclosedherein. See, e.g., U.S. Pat. No. 5,629,193 incorporated herein byreference. A nucleic acid fragment may also have one or more regulatoryelements operably linked thereto.

[0063] The present invention also comprises nucleic acid fragments thatselectively hybridize to mutant desaturase sequences. Such a nucleicacid fragment typically is at least 15 nucleotides in length.Hybridization typically involves Southern analysis (Southern blotting),a method by which the presence of DNA sequences in a target nucleic acidmixture are identified by hybridization to a labeled oligonucleotide orDNA fragment probe. Southern analysis typically involves electrophoreticseparation of DNA digests on agarose gels, denaturation of the DNA afterelectrophoretic separation, and transfer of the DNA to nitrocellulose,nylon, or another suitable membrane support for analysis with aradiolabeled, biotinylated, or enzyme-labeled probe as described insections 9.37-9.52 of Sambrook et al., (1989) Molecular Cloning, secondedition, Cold Spring Harbor Laboratory, Plainview; N.Y.

[0064] A nucleic acid fragment can hybridize under moderate stringencyconditions or, preferably, under high stringency conditions to a mutantdesaturase sequence. High stringency conditions are used to identifynucleic acids that have a high degree of homology to the probe. Highstringency conditions can include the use of low ionic strength and hightemperature for washing, for example, 0.015 M NaCl/0.0015 M sodiumcitrate (0.1×SSC); 0.1% sodium lauryl sulfate (SDS) at 50-65° C.Alternatively, a denaturing agent such as formamide can be employedduring hybridization, e.g., 50% formamide with 0.1% bovine serumalbumin/0.1% Ficoll/0.1% polyvinylpyrrolidone/50 mM sodium phosphatebuffer at pH 6.5 with 750 mM NaCl, 75 mM sodium citrate at 42° C.Another example is the use of 50% formamide, 5×SSC (0.75 M NaCl, 0.075 Msodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodiumpyrophosphate, 5×Denhardt's solution, sonicated salmon sperm DNA (50μg/ml), 0.1% SDS, and 10% dextran sulfate at 42° C., with washes at 42°C. in 0.2×SSC and 0.1% SDS.

[0065] Moderate stringency conditions refers to hybridization conditionsused to identify nucleic acids that have a lower degree of identity tothe probe than do nucleic acids identified under high stringencyconditions. Moderate stringency conditions can include the use of higherionic strength and/or lower temperatures for washing of thehybridization membrane, compared to the ionic strength and temperaturesused for high stringency hybridization. For example, a wash solutioncomprising 0.060 M NaCl/0.0060 M sodium citrate (4×SSC) and 0.1% sodiumlauryl sulfate (SDS) can be used at 50° C., with a last wash in 1×SSC,at 65° C. Alternatively, a hybridization wash in 1×SSC at 37° C. can beused.

[0066] Hybridization can also be done by Northern analysis (Northernblotting), a method used to identify RNAs that hybridize to a knownprobe such as an oligonucleotide, DNA fragment, cDNA or fragmentthereof, or RNA fragment. The probe is labeled with a radioisotope suchas ³²P, by biotinylation or with an enzyme. The RNA to be analyzed canbe usually electrophoretically separated on an agarose or polyacrylamidegel, transferred to nitrocellulose, nylon, or other suitable membrane,and hybridized with the probe, using standard techniques well known inthe art such as those described in sections 7.39-7.52 of Sambrook etal., supra.

[0067] A polypeptide of the invention comprises an isolated polypeptidehaving a mutant amino acid sequence, as well as derivatives and analogsthereof. See, e.g., the mutant amino acid sequences of FIG. 3. By“isolated” is meant a polypeptide that is expressed and produced in anenvironment other than the environment in which the polypeptide isnaturally expressed and produced. For example, a plant polypeptide isisolated when expressed and produced in bacteria or fungi. A polypeptideof the invention also comprises variants of the mutant desaturasepolypeptides disclosed herein, as discussed above.

[0068] In one embodiment of the claimed invention, a plant contains botha delta-12 desaturase mutation and a delta-15 desaturase mutation. Suchplants can have a fatty acid composition comprising very high oleic acidand very low alpha-linolenic acid levels. Mutations in delta-12desaturase and delta-15 desaturase may be combined in a plant by makinga genetic cross between delta-12 desaturase and delta-15 desaturasesingle mutant lines. A plant having a mutation in delta-12 fatty aciddesaturase is crossed or mated with a second plant having a mutation indelta-15 fatty acid desaturase. Seeds produced from the cross areplanted and the resulting plants are selfed in order to obtain progenyseeds. These progeny seeds are then screened in order to identify thoseseeds carrying both mutant genes.

[0069] Alternatively, a line possessing either a delta-12 desaturase ora delta-15 desaturase mutation can be subjected to mutagenesis togenerate a plant or plant line having mutations in both delta-12desaturase and delta-15 desaturase. For example, the IMC 129 line has amutation in the coding region (Glu₁₀₆ to Lys₁₀₆) of the D form of themicrosomal delta-12 desaturase structural gene. Cells (e.g., seeds) ofthis line can be mutagenized to induce a mutation in a delta-15desaturase gene, resulting in a plant or plant line carrying a mutationin a delta-12 fatty acid desaturase gene and a mutation in a delta-15fatty acid desaturase gene.

[0070] Progeny includes descendants of a particular plant or plant line,e.g., seeds developed on an instant plant are descendants. Progeny of aninstant plant include seeds formed on F₁, F₂, F₃, and subsequentgeneration plants, or seeds formed on BC₁, BC₂, BC₃ and subsequentgeneration plants.

[0071] Plants according to the invention preferably contain an alteredfatty acid composition. For example, oil obtained from seeds of suchplants may have from about 69 to about 90% oleic acid, based on thetotal fatty acid composition of the seed. Such oil preferably has fromabout 74 to about 90% oleic acid, more preferably from about 80 to about90%. oleic acid. In some embodiments, oil obtained from seeds producedby plants of the invention may have from about 2.0% to about 5.0%saturated fatty acids, based on total fatty acid composition of theseeds. In some embodiments, oil obtained from seeds of the invention mayhave from about 1.0% to about 14.0% linoleic acid, or from about 0.5% toabout 10.0% α-linolenic acid.

[0072] Oil composition typically is analyzed by crushing and extractingfatty acids from bulk seed samples (e.g., 10 seeds). Fatty acidtriglycerides in the seed are hydrolyzed and converted to fatty acidmethyl esters. Those seeds having an altered fatty acid composition maybe identified by techniques known to the skilled artisan, e.g.,gas-liquid chromatography (GLC) analysis of a bulked seed sample, singleseed or a single half-seed. Half-seed analysis is well known in the artto be useful because the viability of the embryo is maintained and thusthose seeds having a desired fatty acid profile may be planted to formthe next generation. However, half-seed analysis is also known to be aninaccurate representation of genotype of the seed being analyzed. Bulkseed analysis typically yields a more accurate representation of thefatty acid profile of a given genotype. Half-seed analysis of apopulation of seeds is, however, a reliable indicator of the likelihoodof obtaining a desired fatty acid profile. Fatty acid composition canalso be determined on larger samples, e.g., oil obtained by pilot plantor commercial scale refining, bleaching and deodorizing of endogenousoil in the seeds.

[0073] The nucleic acid fragments of the invention can be used asmarkers in plant genetic mapping and plant breeding programs. Suchmarkers may include restriction fragment length polymorphism (RFLP),random amplification polymorphism detection (RAPD), polymerase chainreaction (PCR) or self-sustained sequence replication (3SR) markers, forexample. Marker-assisted breeding techniques may be used to identify andfollow a desired fatty acid composition during the breeding process.Marker-assisted breeding techniques may be used in addition to, or as analternative to, other sorts of identification techniques. An example ofmarker-assisted breeding is the use of PCR primers that specificallyamplify a sequence containing a desired mutation in delta-12 desaturaseor delta-15 desaturase.

[0074] Methods according to the invention are useful in that theresulting plants and plant lines have desirable seed fatty acidcompositions as well as superior agronomic properties compared to knownlines having altered seed fatty acid composition. Superior agronomiccharacteristics include, for example, increased seed germinationpercentage, increased seedling vigor, increased resistance to seedlingfungal diseases (damping off, root rot and the like), increased yield,and improved standability.

[0075] In another aspect, Brassica plants producing seeds having a longchain monounsaturated fatty acid content of at least about 82% and anerucic acid content of at least about 15%, based on total fatty acidcomposition, are featured. As used herein, “long chain” refers to carbonchains of 16 and greater, e.g., chains of 16 to 24 carbons. The longchain monounsaturated fatty acid content is distributed primarily amongoleic acid, eicosenoic acid and erucic acid. The heterogenous nature ofthe long chain monounsaturated fatty acids in the seed oiltriacylglycerols confers desirable properties to the oil.

[0076] High oleic acid lines described herein can be crossed to higherucic acid lines to produce Brassica plants having a high long chainmonounsaturated fatty acid content within their seeds. Suitable higholeic acid lines are described, for example, in Example 5 and Table 17,and have an oleic acid content of about 82% to about 85%, based on totalfatty acid composition. Suitable high erucic acid lines have an erucicacid content of about 45%, based on total fatty acid composition.Brassica plant line HEC01 is a high erucic acid line that isparticularly useful and is sold under the trade name Hero. Other higherucic acid varieties such as Venus, Mercury, Neptune or S89-3673 haveerucic acid contents of about 50% or greater and can also be used.McVetty, P. B. E. et al., Can. J. Plant Sci., 76(2):341-342 (1996);Scarth, R. et al., Can. J. Plant Sci., 75(1):205-206 (1995); andMcVetty, P. B. E. et al., Can. J. Plant Sci., 76(2):343-344 (1996).

[0077] Seeds of the invention have an oleic acid and eicosenoic acidcontent of at least about 37% and 14%, respectively, based on totalfatty acid composition. The total saturated fatty acid content is lessthan about 7%. As used herein, “total saturated fatty acid content”refers to the total of myristate (14:0), palmitate (16:0), stearate(18:0), arachidate (20:0), behenate (22:0) and lignocerate (24:0). Thetotal polyunsaturated content is less than about 11% based on totalfatty acid composition. As used herein, “total polyunsaturated fattyacid content” refers to the sum of linoleic (18:2), α-linolenic (18:3),and eicosadienoic (20:2) fatty acids as a percentage of the total fattyacid content.

[0078] In some embodiments, the monounsaturated content is from about85% to about 90%. The oleic acid content within these seeds is about 42%or greater, and preferably from about 47% to about 56%. The erucic acidand eicosenoic acid content is from about 17% to about 31% and fromabout 15% to about 21%, respectively.

[0079] Seed oils having a long chain monounsaturated content of at leastabout 82% and an erucic acid content of at least about 15%, based ontotal fatty acid composition, are also featured. These oils can beextracted, for example, from a single line of Brassica seeds having asuitable fatty acid composition as described herein. The oleic acid andeicosenoic acid content of these oils is at least about 37% and 14%,respectively, based on total fatty acid composition. The total saturatedand polyunsaturated content of these oils is less than about 7% and 11%,respectively. Preferably, the polyunsaturated content is less than about9%. In some embodiments, the oils have a monounsaturated content of fromabout 85% to about 90%. The oleic acid content of these oils is at leastabout 42% and more preferably, from about 47% to about 56%. The oilshave an erucic acid content of from about 17% to about 31% and aneicosenoic acid content of from about 15% to about 21%.

[0080] Alternatively, it is contemplated that oils of the invention canbe obtained by mixing high-erucic acid rapeseed oil (HEAR) and an oilhaving at least about 87% oleic acid, preferably from about 90% to about95% oleic acid, based on total fatty acid composition. HEAR oil has anerucic acid content of about 49% and an oleic acid content of about 16%.

[0081] Oils having a long chain monounsaturated content of at leastabout 82% unexpectedly have low temperature properties that aredesirable for industrial applications such as lubrication. The basis forthese properties is not known, but is it possible that the heterogeneouschain lengths of the triacylglycerols in oils of the invention impedeorderly packing as the end methyl groups have a mismatch in molecularvolume, reducing Van der Waals interactions. The double bond in eachfatty acid moiety is present at different carbon positions along theacyl chain, which may disrupt packing and also reduce π-π electronicinteractions between adjacent fatty acid chains. The high monounsaturatecontent is thought to provide improved oxidative stability along withhigh fluidity characteristics. The low levels of polyunsaturates in oilsof the invention also promotes high oxidative stability, since the ratesof oxidation of linoleic acid and linolenic acid at 20° C. are 12-20times and 25 times, respectively, larger than the rate of oxidation ofoleic acid.

[0082] Oxidative stability can be measured with an Oxidative StabilityIndex Instrument, Omnion, Inc., Rockland, Mass., according to AOCSOfficial Method Cd 12b-92 (revised 1993). The method is an automatedreplacement for the Active Oxygen Method (AOM) procedure, AOCS OfficialMethod Cd 12-57. Oxidative stability of oils having a long chainmonounsaturated content of at least about 82% is from about 40 AOM hoursto about 100 AOM hours in the absence of added antioxidants. Incomparison, mid-oleic canola oil (about 76% oleic acid) and high erucicacid rapeseed oil have oxidative stabilities of about 38 and 16 AOMhours, respectively, in the absence of added antioxidants.

[0083] The oils of the invention have desirable functional properties,e.g., low temperature behavior and a high viscosity index, along withhigh oxidative stability. The presence of higher molecular weight fattyacids increases the molecular weight of the triacylglycerols, providingthe oil with a higher flash point and a higher fire point. The increasedmolecular weight also improves the viscosity index of the oils.Viscosity index is an arbitrary number that indicates the viscositychange with temperature of a lubricant. The Dean and Davis viscosityindex can be calculated from observed viscosities of a lubricant at 40°C. and 100° C. and can produce values ranging from 0 to values greaterthan 200. A higher viscosity index value indicates that the viscosity ofthe oil changes less with a change in temperature. In other words, thehigher the viscosity index, the smaller the difference in viscositybetween high and low temperatures.

[0084] An oil of the invention can be formulated for industrialapplications such as engine lubricants or as hydraulic fluids byaddition of one or more additives to an oil having a long chainmonounsaturated fatty acid content of at least about 82% and an erucicacid content of at least about 15%, based on total fatty acidcomposition. For example, a transmission fluid for diesel engines can bemade that includes antioxidants, anti-foam additives, anti-wearadditives, corrosion inhibitors, dispersants, detergents, and acidneutralizers, or combinations thereof. Hydraulic oil compositions caninclude antioxidants, anti-rust additives, anti-wear additives, pourpoint depressants, viscosity-index improvers and anti-foam additives orcombinations thereof. Specific formulations will vary depending on theend use of the oil; suitability of a formulation for a specific end usecan be assessed using standard techniques.

[0085] Typical antioxidants include zinc dithiophosphates, methyldithiocarbamates, hindered phenols, phenol sulfides, metal phenolsulfides, metal salicylates, aromatic amines, phospho-sulfurized fatsand olefins, sulfurized olefins, sulfurized fats and fat derivatives,sulfurized paraffins, sulfurized carboxylic acids,disalieylal-1,2,-propane diamine, 2,4-bis(alkyldithio-1,3,4-thiadiazoles) and dilauryl selenide. Antioxidants aretypically present in amounts from about 0.01% to about 5%, based on theweight of the composition. In particular, about 0.01% to about 1.0% ofantioxidant is added to an oil of the invention. See U.S. Pat. No.5,451,334 for additional antioxidants.

[0086] Rust inhibitors protect surfaces against rust and include, forexample, alkylsuccinic type organic acids, and derivatives thereof,alkylthioacetic acids and derivatives thereof, organic amines, organicphosphates, polyhyndric alcohols and sodium and calcium sulphonates.Anti-wear additives adsorb on metal and provide a film that reducesmetal-to-metal contact. In general, anti-wear additives include zincdialkyldithiophosphates, tricresyl phosphate, didodecyl phosphite,sulfurized sperm oil, sulfurized terpenes and zincdialkyldithiocarbamate, and are used in amounts from about 0.05% toabout 4.5%.

[0087] Corrosion inhibitors include dithiophosphates and in particular,zinc dithiophosphates, metal sulfonates, metal phenate sulfides, fattyacids, acid phosphate esters and alkyl succinic acids.

[0088] Pour point depressants permit flow of the oil composition belowthe pour point of the unmodified lubricant. Common pour pointdepressants include polymethacrylates, wax alkylated naphthalenepolymers, wax alkylated phenol polymers and chlorinated polymers and aretypically present in amounts of about 1% or less. See, for example, U.S.Pat. Nos. 5,451,334 and 5,413,725. The viscosity-index can be increasedby adding polyisobutylenes, polymethacrylates, polyacrylates, ethylenepropylene copolymers, styrene isoprene copolymers, styrene butadienecopolymers and styrene maleic ester copolymers.

[0089] Anti-foam additives reduce or prevent the formation of a stablesurface foam and are typically present in amounts from about 0.00003% toabout 0.05%. Polymethylsiloxanes, polymethacrylates, salts of alkylalkylene dithiophosphates, amyl acrylate telomer andpoly(2-ethylhexylacrylate-co-ethyl acrylate are non-limiting examples ofanti-foam additives.

[0090] Detergents and dispersants are polar materials that provide acleaning function. Detergents include metal sulfonates, metalsalicylates and metal thiophosponates. Dispersants include polyaminesuccinimides, hydroxy benzyl polyamines, polyamine succinamides,polyhydroxy succinic esters and polyamine amide imidazolines.

[0091] While the invention is susceptible to various modifications andalternative forms, certain specific embodiments thereof are described inthe general methods and examples set forth below. For example theinvention may be applied to all Brassica species, including B. rapa, B.juncea, and B. hirta, to produce substantially similar results. Itshould be understood, however, that these examples are not intended tolimit the invention to the particular forms disclosed but, instead theinvention is to cover all modifications, equivalents and alternativesfalling within the scope of the invention. This includes the use ofsomaclonal variation; physical or chemical mutagenesis of plant parts;anther, microspore or ovary culture followed by chromosome doubling; orself- or cross-pollination to transmit the fatty acid trait, alone or incombination with other traits, to develop new Brassica lines.

EXAMPLE 1 Mutagenesis

[0092] Seeds of Westar, a Canadian (Brassica napus) spring canolavariety, were subjected to chemical mutagenesis. Westar is a registeredCanadian spring variety with canola quality. The fatty acid compositionof field-grown Westar, 3.9% C_(16:0), 1.9% C_(18:0), 67.5% C_(18:1),17.6% C_(18:2), 7.4% C_(18:3), <2% C20:1+C_(22:1), has remained stableunder commercial production, with ≦±10% deviation, since 1982.

[0093] Prior to mutagenesis, 30,000 seeds of B. napus cv. Westar seedswere preimbibed in 300-seed lots for two hours on wet filter paper tosoften the seed coat. The preimbibed seeds were placed in 80 mMethylmethanesulfonate (EMS) for four hours. Following mutagenesis, theseeds were rinsed three times in distilled water. The seeds were sown in48-well flats containing Pro-Mix. Sixty-eight percent of the mutagenizedseed germinated. The plants were maintained at 25° C./15° C., 14/10 hrday/night conditions in the greenhouse. At flowering, each plant wasindividually self-pollinated.

[0094] M₂ seed from individual plants were individually catalogued andstored, approximately 15,000 M₂ lines was planted in a summer nursery inCarman, Manitoba. The seed from each selfed plant were planted in3-meter rows with 6-inch row spacing. Westar was planted as the checkvariety. Selected lines in the field were selfed by bagging the mainraceme of each plant. At maturity, the selfed plants were individuallyharvested and seeds were catalogued and stored to ensure that the sourceof the seed was known.

[0095] Self-pollinated M₃ seed and Westar controls were analyzed in10-seed bulk samples for fatty acid composition via gas chromatography.Statistical thresholds for each fatty acid component were establishedusing a Z-distribution with a stringency level of 1 in 10,000. Mean andstandard deviation values were determined from the non-mutagenizedWestar control population in the field. The upper and lower statisticalthresholds for each fatty acid were determined from the mean value ofthe population ±the standard deviation, multiplied by theZ-distribution. Based on a population size of 10,000, the confidenceinterval is 99.99%.

[0096] The selected M₃ seeds were planted in the greenhouse along withWestar controls. The seed was sown in 4inch pots containing Pro-Mix soiland the plants were maintained at 25° C./15° C., 14/10 hr day/nightcycle in the greenhouse. At flowering, the terminal raceme wasself-pollinated by bagging. At maturity, selfed M₄ seed was individuallyharvested from each plant, labelled, and stored to ensure that thesource of the seed was known.

[0097] The M₄ seed was analyzed in 10-seed bulk samples. Statisticalthresholds for each fatty acid component were established from 259control samples using a Z-distribution of 1 in 800. Selected M₄ lineswere planted in a field trial in Carman, Manitoba in 3-meter rows with6-inch spacing. Ten M₄ plants in each row were bagged forself-pollination. At maturity, the selfed plants were individuallyharvested and the open pollinated plants in the row were bulk harvested.The M₅ seed from single plant selections was analyzed in 10-seed bulksamples and the bulk row harvest in 50-seed bulk samples.

[0098] Selected M₅ lines were planted in the greenhouse along withWestar controls. The seed was grown as previously described. Atflowering the terminal raceme was self-pollinated by bagging. Atmaturity, selfed M₆ seed was individually harvested from each plant andanalyzed in 10-seed bulk samples for fatty acid composition.

[0099] Selected M₆ lines were entered into field trials in EasternIdaho. The four trial locations were selected for the wide variabilityin growing conditions. The locations included Burley, Tetonia, Lamontand Shelley (Table 7). The lines were planted in four 3-meter rows withan 8-inch spacing, each plot was replicated four times. The plantingdesign was determined using a Randomized Complete Block Design. Thecommercial cultivar Westar was used as a check cultivar. At maturity theplots were harvested to determine yield. Yield of the entries in thetrial was determined by taking the statistical average of the fourreplications. The Least Significant Difference Test was used to rank theentries in the randomized complete block design. TABLE 7 Trial Locationsfor Selected Fatty Acid Mutants LOCATION SITE CHARACTERIZATIONS BURLEYIrrigated. Long season. High temperatures during flowering. TETONIADryland. Short season. Cool temperatures. LAMONT Dryland. Short season.Cool temperatures. SHELLEY Irrigated. Medium season. High temperaturesduring flowering.

[0100] To determine the fatty acid profile of entries, plants in eachplot were bagged for self-pollination. The M₇ seed from single plantswas analyzed for fatty acids in ten-seed bulk samples.

[0101] To determine the genetic relationships of the selected fatty acidmutants crosses were made. Flowers of M₆ or later generation mutationswere used in crossing. F₁ seed was harvested and analyzed for fatty acidcomposition to determine the mode of gene action. The F₁ progeny wereplanted in the greenhouse. The resulting plants were self-pollinated,the F₂ seed harvested and analyzed for fatty acid composition forallelism studies. The F₂ seed and parent line seed was planted in thegreenhouse, individual plants were self-pollinated. The F₃ seed ofindividual plants was tested for fatty acid composition using 10-seedbulk samples as described previously.

[0102] In the analysis of some genetic relationships dihaploidpopulations were made from the microspores of the F₁ hybrids.Self-pollinated seed from dihaploid plants were analyzed for fatty acidanalysis using methods described previously.

[0103] For chemical analysis, 10-seed bulk samples were hand ground witha glass rod in a 15-mL polypropylene tube and extracted in 1.2 mL 0.25 NKOH in 1:1 ether/methanol. The sample was vortexed for 30 sec. andheated for 60 sec. in a 60° C. water bath. Four mL of saturated NaCl and2.4 mL of iso-octane were added, and the mixture was vortexed again.After phase separation, 600 μL of the upper organic phase were pipettedinto individual vials and stored under nitrogen at −5° C. One μL sampleswere injected into a Supelco SP-2330 fused silica capillary column (0.25mm ID, 30 M length, 0.20 μm df).

[0104] The gas chromatograph was set at 180° C. for 5.5 minutes, thenprogrammed for a 2° C./minute increase to 212° C., and held at thistemperature for 1.5 minutes. Total run time was 23 minutes.Chromatography settings were: Column head pressure—15 psi, Column flow(He)—0.7 mL/min., Auxiliary and Column flow—33 mL/min., Hydrogen flow—33mL/min., Air flow—400 mL/min., Injector temperature—250° C., Detectortemperature—300° C., Split vent13 1/15.

[0105] Table 8 describes the upper and lower statistical thresholds foreach fatty acid of interest. TABLE 8 Statistical Thresholds for SpecificFatty Acids Derived from Control Westar Plantings Percent Fatty AcidsGenotype C_(16:0) C_(18:0) C_(18:1) C_(18:2) C_(18:3) Sats* M₃Generation(1 in 10,000 rejection rate) Lower 3.3 1.4 — 13.2 5.3 6.0Upper 4.3 2.5 71.0 21.6 9.9 8.3 M₄ Generation(1 in 800 rejection rate)Lower 3.6 0.8 — 12.2 3.2 5.3 Upper 6.3 3.1 76.0 32.4 9.9 11.2 M₅Generation (1 in 755 rejection rate) Lower 2.7 0.9 —  9.6 2.6 4.5 Upper5.7 2.7 80.3 26.7 9.6 10.0

EXAMPLE 2 High Oleic Acid Canola Lines

[0106] In the studies of Example 1, at the M₃ generation, 31 linesexceeded the upper statistical threshold for oleic acid (≧71.0%). LineW7608.3 had 71.2% oleic acid. At the M₄ generation, its selfed progeny(W7608.3.5, since designated A129.5) continued to exceed the upperstatistical threshold for C_(18:1) with 78.8% oleic acid. M₅ seed offive self-pollinated plants of line A129.5 (ATCC 40811) averaged 75.0%oleic acid. A single plant selection, A129.5.3 had 75.6% oleic acid. Thefatty acid composition of this high oleic acid mutant, which was stableunder both field and greenhouse conditions to the M₇ generation, issummarized in Table 9. This line also stably maintained its mutant fattyacid composition to the M₇ generation in field trials in multiplelocations. Over all locations the self-pollinated plants (A129) averaged78.3% oleic acid. The fatty acid composition of the A129 for each Idahotrial location are summarized in Table 10. In multiple locationreplicated yield trials, A129 was not significantly different in yieldfrom the parent cultivar Westar.

[0107] The canola oil of A129, after commercial processing, was found tohave superior oxidative stability compared to Westar when measured bythe Accelerated Oxygen Method (AOM), American Oil Chemists' SocietyOfficial Method Cd 12-57 for fat stability; Active Oxygen Method(revised 1989). The AOM of Westar was 18 AOM hours and for A129 was 30AOM hours. TABLE 9 Fatty Acid Composition of a High Oleic Acid CanolaLine Produced by Seed Mutagenesis Percent Fatty Acids Genotype C_(16:0)C_(18:0) C_(18:1) C_(18:2) C_(18:3) Sats Westar 3.9 1.9 67.5 17.6 7.47.0 W7608.3 3.9 2.4 71.2 12.7 6.1 7.6 (M₃) W7608.3.5 3.9 2.0 78.8 7.73.9 7.3 (M₄) A129.5.3 3.8 2.3 75.6 9.5 4.9 7.6 (M₅)

[0108] TABLE 10 Fatty Acid Composition of a Mutant High Oleic Acid Lineat Different Field Locations in Idaho Percent Fatty Acids LocationC_(16:0) C_(18:0) C_(18:1) C_(18:2) C_(18:3) Sats Burley 3.3 2.1 77.58.1 6.0 6.5 Tetonia 3.5 3.4 77.8 6.5 4.7 8.5 Lamont 3.4 1.9 77.8 7.4 6.56.3 Shelley 3.3 2.6 80.0 5.7 4.5 7.7

[0109] The genetic relationship of the high oleic acid mutation A129 toother oleic desaturases was demonstrated in crosses made to commercialcanola cultivars and a low linolenic acid mutation. A129 was crossed tothe commercial cultivar Global (C_(16:0)—4.5%, C_(18:0)—1.5%,C_(18:1)—62.9%, C_(18:2)—20.0%, C_(18:3)—7.3%). Approximately 200 F₂individuals were analyzed for fatty acid composition. The results aresummarized in Table 11. The segregation fit 1:2:1 ratio suggesting asingle co-dominant gene controlled the inheritance of the high oleicacid phenotype. TABLE 11 Genetic Studies of A129 X Global C_(18:1)Frequency Genotype Content(%) Observed Expected od − od − 77.3 43 47 od− od + 71.7 106 94 od + od + 66.1 49 47

[0110] A cross between A129 and IMC 01, a low linolenic acid variety(C_(16:0)—4.1%, C_(18:0)—1.9%, C_(18:1)—66.4%, C_(18:2)—18.1%,C_(18:3)—5.7%), was made to determine the inheritance of the oleic aciddesaturase and linoleic acid desaturase. In the F₁ hybrids both theoleic acid and linoleic acid desaturase genes approached the mid-parentvalues indicating a co-dominant gene actions. Fatty acid analysis of theF₂ individuals confirmed a 1:2:1:2:4:2:1:2:1 segregation of twoindependent, co-dominant genes (Table 12). A line was selected from thecross of A129 and IMC01 and designated as IMC130 (ATCC deposit no.75446) as described in U.S. patent application Ser. No. 08/425,108,incorporated herein by reference. TABLE 12 Genetic Studies of A129 X IMC01 Frequency Genotype Ratio Observed Expected od − od − ld − ld − 1 1112 od − od − ld − ld + 2 30 24 od − od − ld + ld + 1 10 12 od − od + ld− ld − 2 25 24 od − od + ld − ld + 4 54 47 od − od + ld + ld + 2 18 24od + od + ld − ld − 1 7 12 od + od + ld − ld + 2 25 24 od + od + ld +ld + 1 8 12

[0111] An additional high oleic acid line, designated A128.3, was alsoproduced by the disclosed method. A 50-seed bulk analysis of this lineshowed the following fatty acid composition: C_(16:0)—3.5%, C_(18:0)131.8%, C_(18:1)—77.3%, C_(18:2)—9.0%, C_(18:3)—5.6%, FDA Sats —5.3%,Total Sats—6.4%. This line also stably maintained its mutant fatty acidcomposition to the M₇ generation. In multiple locations replicated yieldtrials, A128 was not significantly different in yield from the parentcultivar Westar.

[0112] A129 was crossed to A128.3 for allelism studies. Fatty acidcomposition of the F₂ seed showed the two lines to be allelic. Themutational events in A129 and A128.3 although different in origin werein the same gene.

[0113] An additional high oleic acid line, designated M3028.-10 (ATCC75026), was also produced by the disclosed method in Example 1. A10-seed bulk analysis of this line showed the following fatty acidcomposition: C_(16:0)—3.5%, C_(18:0)—1.8%, C_(18:1)—77.3%,C_(18:2)—9.0%, C_(18:3)—5.6%, FDA Saturates—5.3%, Total Saturates—6.4%.In a single location replicated yield trial M3028.10 was notsignificantly different in yield from the parent cultivar Westar.

EXAMPLE 3 Low Linoleic Acid Canola

[0114] In the studies of Example 1, at the M₃ generation, 80 linesexceeded the lower statistical threshold for linoleic acid (≦13.2%).Line W12638.8 had 9.4% linoleic acid. At the M₄ and M₅ generations, itsselfed progenies [W12638.8, since designated A133.1 (ATCC 40812)]continued to exceed the statistical threshold for low C_(18:2) withlinoleic acid levels of 10.2% and 8.4%, respectively. The fatty acidcomposition of this low linoleic acid mutant, which was stable to the M₇generation under both field and greenhouse conditions, is summarized inTable 13. In multiple location replicated yield trials, A133 was notsignificantly different in yield from the parent cultivar Westar. Anadditional low linoleic acid line, designated M3062.8 (ATCC 75025), wasalso produced by the disclosed method. A 10-seed bulk analysis of thisline showed the following fatty acid composition: C_(16:0)—3.8%,C_(18:0)—2.3%, C_(18:1)—77.1%, C_(18:2)—8.9%, C_(18:3)—4.3%, FDASats—6.1%. This line has also stably maintained its mutant fatty acidcomposition in the field and greenhouse. TABLE 13 Fatty Acid Compositionof a Low Linoleic Acid Canola Line Produced by Seed Mutagenesis PercentFatty Acids Genotype C_(16:0) C_(18:0) C_(18:1) C_(18:2) C_(18:3)Sats^(b) Westar 3.9 1.9 67.5 17.6 7.4 7.0 W12638.8 3.9 2.3 75.0 9.4 6.17.5 (M₃) W12638.8.1 4.1 1.7 74.6 10.2 5.9 7.1 (M₄) A133.1.8 3.8 2.0 77.78.4 5.0 7.0 (M₅)

EXAMPLE 4 Low Linolenic and Linoleic Acid Canola

[0115] In the studies of Example 1, at the M₃ generation, 57 linesexceeded the lower statistical threshold for linolenic acid (≦5.3%).Line W14749.8 had 5.3% linolenic acid and 15.0% linoleic acid. At the M₄and M₅ generations, its selfed progenies [W14749.8, since designatedM3032 (ATCC 75021)] continued to exceed the statistical threshold forlow C_(18:3) with linolenic acid levels of 2.7% and 2.3%, respectively,and for a low sum of linolenic and linoleic acids with totals of 11.8%and 12.5% respectively. The fatty acid composition of this low linolenicacid plus linoleic acid mutant, which was stable to the M₅ generationunder both field and greenhouse conditions, is summarized in Table 14.In a single location replicated yield trial M3032 was not significantlydifferent in yield from the parent cultivar (Westar). TABLE 14 FattyAcid Composition of a Low Linolenic Acid Canola Line Produced by SeedMutagenesis Percent Fatty Acids Genotype C_(16:0) C_(18:0) C_(18:1)C_(18:2) C_(18:3) Sats Westar 3.9 1.9 67.5 17.6 7.4 7.0 W14749.8 4.0 2.569.4 15.0 5.3 6.5 (M₃) M3032.8 3.9 2.4 77.9  9.1 2.7 6.4 (M₄) M3032.13.5 2.8 80.0 10.2 2.3 6.5 (M₅)

EXAMPLE 5 Canola Lines Q508 and Q4275

[0116] Seeds of the B. napus line IMC-129 were mutagenized with methylN-nitrosoguanidine (MNNG). The MNNG treatment consisted of three parts:pre-soak, mutagen application, and wash. A 0.05M Sorenson's phosphatebuffer was used to maintain pre-soak and mutagen treatment pH at 6.1.Two hundred seeds were treated at one time on filter paper (Whatman #3M)in a petri dish (100 mm×15 mm). The seeds were pre-soaked in 15 mls of0.05M Sorenson's buffer, pH 6.1, under continued agitation for twohours. At the end of the pre-soak period, the buffer was removed fromthe plate.

[0117] A 10 mM concentration of MNNG in 0.05M Sorenson's buffer, pH 6.1,was prepared prior to use. Fifteen ml of 10 m MNNG was added to theseeds in each plate. The seeds were incubated at 22° C.±3° C. in thedark under constant agitation for four (4) hours. At the end of theincubation period, the mutagen solution was removed.

[0118] The seeds were washed with three changes of distilled water at 10minute intervals. The fourth wash was for thirty minutes. This treatmentregime produced an LD60 population.

[0119] Treated seeds were planted in standard greenhouse potting soiland placed into an environmentally controlled greenhouse. The plantswere grown under sixteen hours of light. At flowering, the racemes werebagged to produce selfed seed. At maturity, the M2 seed was harvested.Each M2 line was given an identifying number. The entire MNNG-treatedseed population was designated as the Q series.

[0120] Harvested M2 seeds was planted in the greenhouse. The growthconditions were maintained as previously described. The racemes werebagged at flowering for selfing. At maturity, the selfed M3 seed washarvested and analyzed for fatty acid composition. For each M3 seedline, approximately 10-15 seeds were analyzed in bulk as described inExample 1.

[0121] High oleic-low linoleic M3 lines were selected from the M3population using a cutoff of >82% oleic acid and <5.0% linoleic. Fromthe first 1600 M3 lines screened for fatty acid composition, Q508 wasidentified. The Q508 M3 generation was advanced to the M4 generation inthe greenhouse. Table 15 shows the fatty acid composition of Q508 andIMC 129. The M4 selfed seed maintained the selected high oleic-lowlinoleic acid phenotype (Table 16). TABLE 15 Fatty Acid Composition ofA129 and High Oleic Acid M3 Mutant Q508 Line# 16:0 18:0 18:1 18:2 18:3A129* 4.0 2.4 77.7 7.8 4.2 Q508 3.9 2.1 84.9 2.4 2.9

[0122] M₄ generation Q508 plants had poor agronomic qualities in thefield compared to Westar. Typical plants were slow growing relative toWestar, lacked early vegetative vigor, were short in stature, tended tobe chlorotic and had short pods. The yield of Q508 was very low comparedto Westar.

[0123] The M₄ generation Q508 plants in the greenhouse tended to bereduced in vigor compared to Westar. However, Q508 yields in thegreenhouse were greater than Q508 yields in the field. TABLE 16 FattyAcid Composition of Seed Oil from Greenhouse-Grown Q508, IMC 129 andWestar. FDA Line 16:0 18:0 18:1 18:2 18:3 Sats IMC 129^(a) 4.0 2.4 77.77.8 4.2 6.4 Westar^(b) 3.9 1.9 67.5 17.6 7.4 >5.8 Q508^(c) 3.9 2.1 84.92.4 2.9 6.0

[0124] Nine other M4 high-oleic low-linoleic lines were also identified:Q3603, Q3733, Q4249, Q6284, Q6601, Q6761, Q7415, Q4275, and Q6676. Someof these lines had good agrononic characteristics and an elevated oleicacid level in seeds of about 80% to about 84%.

[0125] Q4275 was crossed to the variety Cyclone. After selfing for sevengenerations, mature seed was harvested from 93GS34-179, a progeny lineof the Q4275xCyclone cross. Referring to Table 17, fatty acidcomposition of a bulk seed sample shows that 93GS34 retained the seedfatty acid composition of Q4275. 93GS34-179 also maintainedagronomically desirable characteristics.

[0126] After more than seven generations of selfing of Q4275, plants ofQ4275, IMC 129 and 93GS34 were field grown during the summer season. Theselections were tested in 4 replicated plots (5 feet×20 feet) in arandomized block design. Plants were open pollinated. No selfed seed wasproduced. Each plot was harvested at maturity, and a sample of the bulkharvested seed from each line was analyzed for fatty acid composition asdescribed above. The fatty acid compositions of the selected lines areshown in Table 17. TABLE 17 Fatty Acid Composition of Field Grown IMC129, Q4275 and 93GS34 Seeds Fatty Acid Composition (%) Line C_(16:0)C_(18:0) C_(18:1) C_(18:2) C_(18:3) FDA Sats IMC 129 3.3 2.4 76.7 8.75.2 5.7 Q4275 3.7 3.1 82.1 4.0 3.5 6.8 93GS34-179 2.6 2.7 85.0 2.8 3.35.3

[0127] The results shown in Table 17 show that Q4275 maintained theselected high oleic-low linoleic acid phenotype under field conditions.The agronomic characteristics of Q4275 plants were superior to those ofQ508.

[0128] M₄ generation Q508 plants were crossed to a dihaploid selectionof Westar, with Westar serving as the female parent. The resulting F1seed was termed the 92EF population. About 126 F1 individuals thatappeared to have better agronomic characteristics than the Q508 parentwere selected for selfing. A portion of the F₂ seed from suchindividuals was replanted in the field. Each F2 plant was selfed and aportion of the resulting F3 seed was analyzed for fatty acidcomposition. The content of oleic acid in F₃ seed ranged from 59 to 79%.No high oleic (>80%) individuals were recovered with good agronomictype.

[0129] A portion of the F₂ seed of the 92EF population was planted inthe greenhouse to analyze the genetics of the Q508 line. F₃ seed wasanalyzed from 380 F2 individuals. The C_(18:1) levels of F₃ seed fromthe greenhouse experiment is depicted in FIG. 1. The data were testedagainst the hypothesis that Q508 contains two mutant genes that aresemi-dominant and additive: the original IMC 129 mutation as well as oneadditional mutation. The hypothesis also assumes that homozygous Q508has greater than 85% oleic acid and homozygous Westar has 62-67% oleicacid. The possible genotypes at each gene in a cross of Q508 by Westarmay be designated as: AA = Westar Fad2^(a) BB = Westar Fad2^(b) aa =Q508 Fad2^(a−) bb = Q508 Fad2^(b−)

[0130] Assuming independent segregation, a 1:4:6:4:1 ratio of phenotypesis expected. The phenotypes of heterozygous plants are assumed to beindistinguishable and, thus, the data were tested for fit to a 1:14:1ratio of homozygous Westar: heterozygous plants: homozygous Q508.Phenotypic # of Ratio Westar Alleles Genotype 1 4 AABB(Westar) 4 3 AABb,AaBB, AABb, AaBB 6 2 AaBb, AAbb, AaBb, AaBb, aaBB, AaBb 4 1 Aabb, aaBb,Aabb, aaBb 1 0 aabb (Q508)

[0131] Using Chi-square analysis, the oleic acid data fit a 1:14:1ratio. It was concluded that Q508 differs from Westar by two major genesthat are semi-dominant and additive and that segregate independently. Bycomparison, the genotype of IMC 129 is aaBB.

[0132] The fatty acid composition of representative F3 individualshaving greater than 85% oleic acid in seed oil is shown in Table 18. Thelevels of saturated fatty acids are seen to be decreased in such plants,compared to Westar. TABLE 18 92EF F₃ Individuals with >85% C_(18:1) inSeed Oil F3 Plant Fatty Acid Composition (%) Identifier C16:0 C18:0C18:1 C18:2 C18:3 FDASA +38068 3.401 1.582 85.452 2.134 3.615 4.983+38156 3.388 1.379 85.434 2.143 3.701 4.767 +38171 3.588 1.511 85.2892.367 3.425 5.099 +38181 3.75 1.16 85.312 2.968 3.819 4.977 +38182 3.5290.985 85.905 2.614 3.926 4.56 +38191 3.364 1.039 85.737 2.869 4.0394.459 +38196 3.557 1.182 85.054 2.962 4.252 4.739 +38202 3.554 1.10586.091 2.651 3.721 4.713 +38220 3.093 1.16 86.421 1.931 3.514 4.314+38236 3.308 1.349 85.425 2.37 3.605 4.718 +38408 3.617 1.607 85.34 2.333.562 5.224 +38427 3.494 1.454 85.924 2.206 3.289 4.948 +38533 3.641.319 85.962 2.715 3.516 4.959

EXAMPLE 6 Leaf and Root Fatty Acid Profiles of Canola Lines IMC-129,Q508, and Westar

[0133] Plants of Q508, IMC 129 and Westar were grown in the greenhouse.Mature leaves, primary expanding leaves, petioles and roots wereharvested at the 6-8 leaf stage, frozen in liquid nitrogen and stored at−70° C. Lipid extracts were analyzed by GLC as described in Example 1.The fatty acid profile data are shown in Table 19. The data in Table 19indicate that total leaf lipids in Q508 are higher in C_(18:1) contentthan the C_(18:2) plus C_(18:3) content. The reverse is true for Westarand IMC 129. The difference in total leaf lipids between Q508 and IMC129 is consistent with the hypothesis that a second Fad2 gene is mutatedin Q508.

[0134] The C_(16:3) content in the total lipid fraction was about thesame for all three lines, suggesting that the plastid FadC gene productwas not affected by the Q508 mutations. To confirm that the FadC genewas not mutated, chloroplast lipids were separated and analyzed. Nochanges in chioroplast C_(16:1), C_(16:2) or C_(16:3) fatty acids weredetected in the three lines. The similarity in plastid leaf lipids amongQ508, Westar and IMC 129 is consistent with the hypothesis that thesecond mutation in Q508 affects a microsomal Fad2 gene and not a plastidFadC gene. TABLE 19 MATURE EXPANDING LEAF LEAF PETIOLE ROOT West. 1293Q508 West. 129 3Q508 West. 129 3Q508 West. 129 3Q508 16:0 12.1 11.910.1 16.4 16.1 11.3 21.7 23.5 11.9 21.1 21.9 12.0 16:1 0.8 0.6 1.1 0.70.6 1.1 1.0 1.3 1.4 — — — 16:2 2.3 2.2 2.0 2.8 3.1 2.8 1.8 2.2 1.8 — — —16:3 14.7 15.0 14.0 6.3 5.4 6.9 5.7 4.6 5.7 — — — 18:0 2.2 1.6 1.2 2.52.8 1.5 3.7 4.0 1.6  3.6  2.9  2.5 18:1 2.8 4.9 16.7 3.8 8.3 38.0 4.912.9 46.9  3.5  6.1 68.8 18:2 12.6 11.5 6.8 13.3 13.8 4.9 20.7 18.3 5.228.0 30.4  4.4 18:3 50.6 50.3 46.0 54.2 50.0 33.5 40.4 33.2 25.3 43.838.7 12.3

EXAMPLE 7 Sequences of Mutant and Wild-Type Delta-12 Fatty AcidDesaturases from B. napus

[0135] Primers specific for the FAD2 structural gene were used to clonethe entire open reading frame (ORF) of the D and F delta-12 desaturasegenes by reverse transcriptase polymerase chain reaction (RT-PCR). RNAfrom seeds of IMC 129, Q508 and Westar plants was isolated by standardmethods and was used as template. The RT-amplified fragments were usedfor nucleotide sequence determination. The DNA sequence of each genefrom each line was determined from both strands by standard dideoxysequencing methods.

[0136] Sequence analysis revealed a G to A transversion at nucleotide316 (from the translation initiation codon) of the D gene in both IMC129 and Q508, compared to the sequence of Westar. The transversionchanges the codon at this position from GAG to AAG and results in anon-conservative substitution of glutamic acid, an acidic residue, forlysine a basic residue. The presence of the same mutation in both lineswas expected since the Q508 line was derived from IMC 129. The same basechange was also detected in Q508 and IMC 129 when RNA from leaf tissuewas used as template.

[0137] The G to A mutation at nucleotide 316 was confirmed by sequencingseveral independent clones containing fragments amplified directly fromgenomic DNA of IMC 129 and Westar. These results eliminated thepossibility of a rare mutation introduced during reverse transcriptionand PCR in the RT-PCR protocol. It was concluded that the IMC 129 mutantis due to a single base transversion at nucleotide 316 in the codingregion of the D gene of rapeseed microsomal delta 12-desaturase.

[0138] A single base transition from T to A at nucleotide 515 of the Fgene was detected in Q508 compared to the Westar sequence. The mutationchanges the codon at this position from CTC to CAC, resulting in thenon-conservative substitution of a non-polar residue, leucine, for apolar residue, histidine, in the resulting gene product. No mutationswere found in the F gene sequence of IMC 129 compared to the F genesequence of Westar.

[0139] These data support the conclusion that a mutation in a delta-12desaturase gene sequence results in alterations in the fatty acidprofile of plants containing such a mutated gene. Moreover, the datashow that when a plant line or species contains two delta-12 desaturaseloci, the fatty acid profile of an individual having two mutated locidiffers from the fatty acid profile of an individual having one mutatedlocus.

[0140] The mutation in the D gene of IMC 129 and Q508 mapped to a regionhaving a conserved amino acid motif (His-Xaa-Xaa-Xaa-His) found incloned delta-12 and delta-15 membrane bound-desaturases (Table 20).TABLE 20 Alignment of Amino Acid Sequences of Cloned Canola MembraneBound-Desaturases Desaturase Gene Sequence^(a) Position Canola-fad2-D(mutant) AHKCGH 109-114 Canola-Fad2-D AHECGH 109-114 Canola-Fad2-FAHECGH 109-114 Canola-FadC GHDC A H 170-175 Canola-fad3 (mutant) GHKCGH 94-99 Canola-Fad3 GHDCGH  94-99 Canola-FadD GHDCGH 125-130

EXAMPLE 8 Transcription and Translation of Microsomal Delta-12 FattyAcid Desaturases

[0141] Transcription in vivo was analyzed by RT-PCR analysis of stage IIand stage III developing seeds and leaf tissue. The primers used tospecifically amplify delta-12 desaturase F gene RNA from the indicatedtissues were sense primer 5′-GGATATGATGATGGTGAAAGA-3′ and antisenseprimer 5′-TCTTTCACCATCATCATATCC-3′. The primers used to specificallyamplify delta-12 desaturase D gene RNA from the indicated tissues weresense primer 5′-GTTATGAAGCAAAGAAGAAAC-3′ and antisense primer5′-GTTTCTTCTTTGCTTCATAAC-3′. The results indicated that mRNA of both theD and F gene was expressed in seed and leaf tissues of IMC 129, Q508 andwild type Westar plants.

[0142] In vitro transcription and translation analysis showed that apeptide of about 46 kD was made. This is the expected size of both the Dgene product and the F gene product, based on sum of the deduced aminoacid sequence of each gene and the cotranslational addition of amicrosomal membrane peptide.

[0143] These results rule out the possibility that non-sense orframeshift mutations, resulting in a truncated polypeptide gene product,are present in either the mutant D gene or the mutant F gene. The data,in conjunction with the data of Example 7, support the conclusion thatthe mutations in Q508 and IMC 129 are in delta-12 fatty acid desaturasestructural genes encoding desaturase enzymes, rather than in regulatorygenes.

EXAMPLE 9 Development of Gene-Specific PCR Markers

[0144] Based on the single base change in the mutant D gene of IMC 129described in above, two 5′ PCR primers were designed. The nucleotidesequence of the primers differed only in the base (G for Westar and Afor IMC 129) at the 3′ end. The primers allow one to distinguish betweenmutant fad2-D and wild-type Fad2-D alleles in a DNA-based PCR assay.Since there is only a single base difference in the 5′ PCR primers, thePCR assay is very sensitive to the PCR conditions such as annealingtemperature, cycle number, amount, and purity of DNA templates used.Assay conditions have been established that distinguish between themutant gene and the wild type gene using genomic DNA from IMC 129 andwild type plants as templates. Conditions may be further optimized byvarying PCR parameters, particularly with variable crude DNA samples. APCR assay distinguishing the single base mutation in IMC 129 from thewild type gene along with fatty acid composition analysis provides ameans to simplify segregation and selection analysis of genetic crossesinvolving plants having a delta-12 fatty acid desaturase mutation.

EXAMPLE 10 Transformation with Mutant and Wild Type Fad3 Genes

[0145]B. napus cultivar Westar was transformed with mutant and wild typeFad3 genes to demonstrate that the mutant Fad3 gene for canolacytoplasmic linoleic desaturase delta-15 desaturase is nonfunctional.Transformation and regeneration were performed using disarmedAgrobacterium tumefaciens essentially following the procedure describedin WO 94/11516.

[0146] Two disarmed Agrobacterium strains were engineered, eachcontaining a Ti plasmid having the appropriate gene linked to aseed-specific promoter and a corresponding termination sequence. Thefirst plasmid, pIMC110, was prepared by inserting into a disarmed Tivector the full length wild type Fad3 gene in sense orientation(nucleotides 208 to 1336 of SEQ ID 6 in WO 93/11245), flanked by a napinpromoter sequence positioned 5′ to the Fad3 gene and a napin terminationsequence positioned 3′ to the Fad3 gene. The rapeseed napin promoter isdescribed in EP 0255378.

[0147] The second plasmid, pIMC205, was prepared by inserting a mutatedFad3 gene in sense orientation into a disarmed Ti vector. The mutantsequence contained mutations at nucleotides 411 and 413 of themicrosomal Fad3 gene described in WO93/11245, thus changing the sequencefor codon 96 from GAC to AAG. The amino acid at codon 96 of the geneproduct was thereby changed from aspartic acid to lysine. See Table 20.A bean (Phaseolus vulgaris) phaseolin (7S seed storage protein) promoterfragment of 495 base pairs was placed 5′ to the mutant Fad3 gene and aphaseolin termination sequence was placed 3′ to the mutant Fad3 gene.The phaseolin sequence is described in Doyle et al., (1986) J. Biol.Chem. 261:9228-9238) and Slightom et al., (1983) Proc. Natl. Acad. Sci.USA 80:1897-1901.

[0148] The appropriate plasmids were engineered and transferredseparately to Agrobacterium strain LBA4404. Each engineered strain wasused to infect 5 mm segments of hypocotyl explants from Westar seeds bycocultivation. Infected hypocotyls were transferred to callus mediumand, subsequently, to regeneration medium. Once discernable stems formedfrom the callus, shoots were excised and transferred to elongationmedium. The elongated shoots were cut, dipped in Rootone™, rooted on anagar medium and transplanted to potting soil to obtain fertile T1plants. T2 seeds were obtained by selfing the resulting T1 plants.

[0149] Fatty acid analysis of T2 seeds was carried out as describedabove. The results are summarized in Table 21. Of the 40 transformantsobtained using the pIMC 110 plasmid, 17 plants demonstrated wild typefatty acid profiles and 16 demonstrated overexpression. A proportion ofthe transformants are expected to display an overexpression phenotypewhen a functioning gene is transformed in sense orientation into plants.

[0150] Of the 307 transformed plants having the pIMC205 gene, noneexhibited a fatty acid composition indicative of overexpression. Thisresult indicates that the mutant fad3 gene product is non-functional,since some of the transformants would have exhibited an overexpressionphenotype if the gene product were functional. TABLE 21 Overexpressionand Co-suppression Events in Westar Populations Transformed with pIMC205or pIMC110. Number of α-Linolenic Overexpression Co-Suppression WildTrans- Acid Events Events Type Construct formants Range (%) (>10%linolenic) (<4.0% linolenic) Events pIMC110 40 2.4-20.6 16 7 17 pIMC205307 4.6-10.4 0 0 307

[0151] Fatty acid compositions of representative transformed plants arepresented in Table 22. Lines 652-09 and 663-40 are representative ofplants containing pIMC110 and exhibiting an overexpression and aco-suppression phenotype, respectively. Line 205-284 is representativeof plants containing pIMC205 and having the mutant fad3 gene. TABLE 22Fatty Acid Composition of T2 Seed From Westar Transformed With pIMC205or pIMC110. Fatty Acid Composition (%) Line C16:0 C18:0 C18:1 C18:2C18:3 652-09 pIMC110 4.7 3.3 65.6 8.1 14.8 overexpression 663-40 4.9 2.162.5 23.2 3.6 pIMC110 co-suppression 205-284 3.7 1.8 68.8 15.9 6.7pIMC205

EXAMPLE 11 Sequences of Wild Type and Mutant Fad2-D and Fad2-F

[0152] High molecular weight genomic DNA was isolated from leaves ofQ4275 plants (Example 5). This DNA was used as template foramplification of Fad2-D and Fad2-F genes by polymerase chain reaction(PCR). PCR amplifications were carried out in a total volume of 100 μland contained 0.3 μg genomic DNA, 200 μM deoxyribonucleosidetriphosphates, 3 mM MgSO₄, 1-2 Units DNA polymerase and 1×Buffer(supplied by the DNA polymerase manufacturer). Cycle conditions were: 1cycle for 1 min at 95° C., followed by 30 cycles of 1 min at 94° C., 2min at 55° C. and 3 min at 73° C.

[0153] The Fad2-D gene was amplified once using Elongase® (Gibco-BRL).PCR primers were: CAUCAUCAUCAUCTTCTTCGTAGGGTTCATCG andCUACUACUACUATCATAGAAGAGAAAGGTTCAG for the 5′ and 3′ ends of the gene,respectively.

[0154] The Fad2-F gene was independently amplified 4 times, twice withElongase® and twice with Taq polymerase (Boehringer Mannheim). The PCRprimers used were: 5′CAUCAUCAUCAUCATGGGTGCACGTGGAAGAA3′ and5′CUACUACUACUATCTTTCACCATCATCATATCC3′ for the 5′ and 3′ ends of thegene, respectively.

[0155] Amplified DNA products were resolved on an agarose gel, purifiedby JetSorb® and then annealed into pAMP1 (Gibco-BRL) via the (CAU)₄ and(CUA)₄ sequences at the ends of the primers, and transformed into E.coli DH5α.

[0156] The Fad2-D and Fad2-F inserts were sequenced on both strands withan ABI PRISM 310 automated sequencer (Perkin-Elmer) following themanufacturer's directions, using synthetic primers, AmpliTaq® DNApolymerase and dye terminator.

[0157] The Fad2-D gene was found to have an intron upstream of the ATGstart codon. As expected, the coding sequence of the gene contained a Gto A mutation at nucleotide 316, derived from IMC 129 (FIG. 2).

[0158] A single base transversion from G to A at nucleotide 908 wasdetected in the F gene sequence of the Q4275 amplified products,compared to the wild type F gene sequence (FIG. 2). This mutationchanges the codon at amino acid 303 from GGA to GAA, resulting in thenon-conservative substitution of a glutamic acid residue for a glycineresidue (Table 3 and FIG. 3). Expression of the mutant Q4275 Fad2-Fdelta-12 desaturase gene in plants alters the fatty acid composition, asdescribed hereinabove.

EXAMPLE 12 High Erucic, High Oleic Acid Rapeseed

[0159] The breeding procedure designed to produce novel fatty acidcompositions in rapeseed is outlined in FIG. 4. In general crosses weremade between a high erucic acid line and a high oleic acid line. Thehigh erucic acid line, designated HECO1 (sold under the trade nameHero), contains about 45.5% erucic acid (Table 23). The high oleic acidlines were designated 93GS66A-130 and 93GS34A-179 and were derived from93GS. See, for example, Example 5 and Table 17. These lines containabout 84% oleic acid in their seed oil (Table 24). TABLE 23 Fatty AcidComposition of HEC01 Fatty Acid Weight (%) C_(14:0) 0.05 C_(16:0) 3.60C_(16:1) 0.36 C_(18:0) 1.66 C_(18:1) 14.72 C_(18:2) 10.67 C_(18:3) 9.71C_(20:0) 1.36 C_(20:1) 9.04 C_(20:2) 0.48 C_(22:0) 1.74 C_(22:1) 45.45C_(24:0) 0.49 C_(24:1) 0.81

[0160] TABLE 24 Fatty Acid Composition of 93GS66A-130 and 93GS34A-179Fatty Acid Weight(%) of 93GS66A-130 Weight(%) of 93GS34A-179 C_(14:0)0.04 0.05 C_(16:0) 3.25 3.23 C_(16:1) 0.25 0.25 C_(18:0) 1.60 1.94C_(18:1) 84.38 83.71 C_(18:2) 2.58 3.14 C_(18:3) 4.86 4.76 C_(20:0) 0.560.65 C_(20:1) 1.57 1.41 C_(20:2) 0.05 0.04 C_(22:0) 0.37 0.39 C_(22:1)0.06 0.03 C_(24:0) 0.20 0.18 C_(24:1) 0.21 0.18

[0161] The F₁ generations of crosses between HEC01×93GS66A-130, andHEC01×93GS34A-179, were designated 96.801 and 96.804, respectively. F₁96.801 and 96.804 plants were self-pollinated to produce F₂ seed.Overall, 622 random single F₂ seeds were analyzed for their fatty acidcomposition. Table 25 summarizes the average percent and standarddeviation for total monounsaturated content, oleic acid, eicosenoicacid, erucic acid, total polyunsaturated and total saturated fatty acidcontent of these 622 seeds. TABLE 25 Fatty acid % Total long chain 78.90± 4.07 monounsaturated Palmitoleate  0.28 ± 0.06 Oleic Acid 45.33 ± 9.91Eicosenoic Acid 14.84 ± 2.84 Erucic Acid 17.97 ± 8.9  Nervonic Acid 0.48 ± 0.21 Total polyunsaturated  7.10 ± 1.05 Total saturated 13.99 ±3.83

[0162] Analysis of this data indicate that the frequency distributionsdeviate from a normal distribution. The total long chain monounsaturatedcontent frequency distribution is slightly skewed to the right(−0.0513), and the eicosenoic acid content distribution is stronglyskewed to the right (−1.715). Frequency distributions for oleic acid anderucic acid content are strongly skewed to the left (0.397 and 0.177,respectively). Skewness was calculated using Lotus 1-2-3 for Windows(release 5.0).

[0163] Table 26 describes characteristics of selected populations withinthe total population of seeds. For example, 151 seeds had a long chainmonounsaturated fatty acid content greater than 82% (Table 26, columnB). Within this population, the average oleic, eicosenoic and erucicacid content was about 48%, 16%, and 19%, respectively. Totalpolyunsaturated fatty acid content (C18:2, C18:3, and C20:2) was about9% and total saturated fatty acid content was less than 7%.

[0164] Forty-seven of the 622 seeds had a long chain monounsaturatedcontent greater than 85% (Table 26, column C). The average oleic,eicosenoic and erucic acid content within these seeds was 51%, 17%, and17%, respectively. Total saturated and total polyunsaturated fatty acidswere each less than 7%.

[0165] Twenty-three of the seeds had an eicosenoic acid content greaterthan 19°/(Table 26, column F). Within these seeds, the average oleicacid erucic acid content was about 44% and 19%, respectively. Totalpolyunsaturated fatty acids were less than 10% and total saturated fattyacids were less than 7%. TABLE 26 A B C D E F Total Saturated 6.76 ±0.72 6.65 ± 0.07 6.68 ± 0.61 6.85 ± 0.98 6.85 ± 0.93 6.66 ± 0.78 C14:00.04 ± 0.04 0.07 ± 0.05 0.06 ± 0.03 0.07 ± 0.04 0.06 ± 0.64 0.07 ± 0.05C16:0 3.42 ± 0.37 3.35 ± 0.34 3.28 ± 0.32 3.45 ± 0.40 3.51 ± 0.42 3.31 ±0.33 C18:0 1.92 ± 0.33 1.93 ± 0.32 2.06 ± 0.32 1.83 ± 0.30 1.90 ± 0.301.93 ± 0.23 C20:0 0.77 ± 0.14 0.76 ± 0.14 0.76 ± 0.13 0.80 ± 0.13 0.76 ±0.12 0.79 ± 0.16 C22:0 0.38 ± 0.14 0.36 ± 0.12 0.37 ± 0.11 0.42 ± 0.150.35 ± 0.12 0.35 ± 0.29 C24:0 0.21 ± 0.19 0.19 ± 0.14 0.20 ± 0.17 0.28 ±0.76 0.26 ± 0.71 0.19 ± 0.24 Total Monounsaturated 82.91 ± 2.11  84.21 ±1.64  86.21 ± 1.00  79.49 ± 4.00  80.36 ± 3.75  83.92 ± 2.43  C16:1 0.28± 0.05 0.27 ± 0.05 0.27 ± 0.04 0.27 ± 0.05 0.27 ± 0.05 0.26 ± 0.05 C18:146.45 ± 9.47  47.66 ± 9.22  51.33 ± 8.96  39.29 ± 6.21  43.55 ± 6.91 44.08 ± 2.89  C20:1 16.91 ± 2.57  16.41 ± 2.47  16.72 ± 2.40  15.56 ±2.22  16.78 ± 1.30  19.97 ± 0.63  C22:1 19.69 ± 8.45  19.38 ± 8.25 17.39 ± 12.26 23.81 ± 5.89  19.27 ± 6.39  19.09 ± 2.32  C24:1 0.49 ±0.19 0.48 ± 0.17 0.50 ± 0.18 0.56 ± 0.19 0.49 ± 0.21 0.52 ± 0.29 TotalPolyunsaturated 10.33 ± 2.10  9.14 ± 1.71 7.11 ± 0.98 13.66 ± 3.87 12.80 ± 3.60  9.43 ± 2.43 C18:2 5.16 ± 1.5  4.36 ± 1.24 3.17 ± 0.82 7.25± 2.62 6.58 ± 2.40 3.96 ± 1.39 C18:3 5.02 ± 1.18 4.65 ± 1.07 3.84 ± 0.696.19 ± 1.61 6.02 ± 1.52 5.27 ± 1.25 C20:2 0.15 ± 0.10 0.13 ± 0.04 0.10 ±0.04 0.22 ± 0.11 0.19 ± 0.10 0.20 ± 0.28

[0166] Fatty acid composition of selected single seeds is presented inTable 27. V800655.334 was a single seed that had a long chainmonounsaturated fatty acid content of approximately 84%. The oleicacid,. eicosenoic acid and erucic acid content was 33.48%, 17.14%, and32.23%, respectively. The total polyunsaturated fatty acid content wasapproximately 10%. The linoleic, α-linolenic and erucic acid content was3.54%, 6.01%, and 0.15%, respectively.

[0167] V800655.126 was a single seed that had a long chainmonounsaturated fatty acid content of approximately 85% (42.67% oleicacid, 16.21% eicosenoic acid, and 25.37% erucic acid). The totalpolyunsaturated fatty acid content was approximately 8% (4.87% linoleicacid, 3.05% α-linolenic acid, and 0.13% eicosadienoic acid).

[0168] V800654.9 was a single seed that had a long chain monounsaturatedfatty acid content of 89% (51.53% oleic acid, 16.94% eicosenoic acid,and 19.24% erucic acid). The total polyunsaturated fatty acid contentwas approximately 8% (4.87% linoleic acid, 3.05% α-linolenic acid, and0.13% eicosadienoic acid).

[0169] Single seeds having a long chain monounsaturated fatty acidcontent of at least about 82% and an erucic acid content of at leastabout 15% were planted in a greenhouse, grown to maturity andself-pollinated. Seed (F₃ generation) from each plant were harvested. Abulk seed sample from each F₂ plant is analyzed for fatty acidcomposition. TABLE 27 Fatty Acid Composition of Selected Single SeedsV800655.334 V800655.126 V800654.9 Fatty Acid Weight (%) Weight (%)Weight (%) C_(14:0) 0.07 0.05 0.03 C_(16:0) 3.49 3.52 2.98 C_(16:1) 0.340.28 0.28 C_(18:0) 1.64 1.89 1.65 C_(18:1) 33.48 42.67 51.53 C_(18:2)3.54 4.87 2.09 C_(18:3) 6.01 3.05 3.53 C_(20:0) 0.86 0.87 0.68 C_(20:1)17.14 16.21 16.94 C_(20:2) 0.15 0.13 0.10 C_(22:0) 0.41 0.35 0.24C_(22:1) 32.23 25.37 19.24 C_(24:0) 0.12 0.13 0.14 C_(24:1) 0.52 0.610.59

[0170] Additional crosses were made between Hero and several high oleiclines (Table 28) to increase the seed erucic acid content through areduction in polyunsaturates content and increase in totalmonunsaturates content. The high oleic acid lines included 048X058 andQ4275X663-40. The 048X058 line resulted from a cross of two separatetransformed lines. The 048X058 line contains a co-suppression eventresulting from introduction of the 663-40 transgene described above, anda second co-suppression event resulting from a transgene that includesan oleosin promoter linked to an oleic desaturase gene. The Q4275X663-40line was derived from a cross of Q4275 (Example 5 and Table 17) by663-40. The 663-40 line contains a co-suppression event resulting from atransgene that includes a napin promotor linked to a linoleic desaturasegene. Plants of each line were grown in growth chambers under 16 hrs oflight at 23/17° C. day/night temperature. Flowers were emasculated priorto opening and covered to prevent cross pollination. On the followingday, stigmas of emasulated flowers were pollinated with the desiredpollen donor. At pod maturity the F1 seed was harvested. TABLE 28 Higherucic crossing block Source of Cross Female Female Male Male MaleNumber Parent Phenotype Parent Phenotype Phenotype 97HEHOA HE101 High22:1 048 × 058 High 18:1/ Transgenes Low 18:3 97HEHOB HE101 High 22:1Q4275 × 663 − 40 High 18:1/ Mutant/ Low 18:3 Transgene 97HEHOC HE101High 22:1 Q4275 × 663 − 40 High 18:1/ Mutant/ Low 18:3 Transgene

[0171] F1 seed generated from the crosses in Table 28 were advanced toF2 seed generation in the growth chamber. Ten seeds were individuallyplanted for each cross. At flowering the plants were covered with bagsto ensure self pollination. The F2 seeds were harvested at maturity.

[0172] The seeds were germinated on filter paper at room temperature inthe dark. Eighteen to 24 hours after the start of germination, onecotyledon was removed from the seed for extraction of fatty acids. Fattyacid compositions were determined using gas chromatography. Selected F2half seeds having a high erucic content are shown in Tables 29 and 30.TABLE 29 Half Seed Selection on F2 Seed of 97HEHOA [HE101 × (048 × 052)]Fatty Acid Composition (%) Sample No. C16:0 C18:0 C18:1 C18:2 C18:3C20:0 C20:1 C22:0 C22:1 C24:0 C24:1 VL10186-1 3.34 1.83 49.7 3.32 1.590.87 19.62 0.30 18.10 0.39 0.35 VL10186-5 2.61 1.07 29.14 5.81 2.42 0.7114.99 0.31 40.90 0.93 0.60 VL10186-33 3.47 1.32 29.73 4.38 2.98 0.8612.22 0.44 41.21 1.50 1.28 VL10186-59 4.01 1.68 41.38 2.96 1.69 1.0819.72 0.43 26.05 0 0.55 VL10186-67 3.90 1.29 29.10 3.65 2.89 0.88 13.790.52 40.96 1.31 1.09 VL10186-74 2.76 1.25 34.04 2.63 1.45 0.75 16.640.38 38.67 0.14 0.95 VL10186-88 3.23 1.39 48.97 3.27 1.50 0.60 19.710.17 20.48 0 0.36

[0173] TABLE 30 Half Seed Selection on F2 Seed of 97HEHOC [HE101 ×(Q4275 × 663 − 40)] Fatty Acid Composition (%) Sample No. C16:0 C18:0C18:1 C18:2 C18:3 C20:0 C20:1 C22:0 C22:1 C24:0 C24:1 VL10200-214 2.240.74 31.66 3.01 6.24 0.46 11.79 0.41 40.60 0.86 1.57 VL10200-231 3.891.03 31.51 12.50 2.41 0.54 14.17 0.29 32.23 0 0.75 VL10200-238 3.36 0.9533.19 8.99 1.66 0.55 14.35 0.21 33.61 0.83 1.02 VL10200-267 3.12 1.0230.18 7.61 1.52 0.59 14.53 0.19 39.41 0.24 1.013 VL10203-50 2.63 0.9731.79 8.47 1.99 0.58 14.58 0.25 37.41 0.13 0.59 VL10200-293 2.71 0.7832.83 6.85 1.88 0.46 13.11 0.32 39.18 0.82 0.67

[0174] Selected half seeds were planted in soil and grown under growthchamber conditions described above. At flowering the plants were coveredwith bags for self pollination. After maturity, the F3 selfed seed washarvested and analyzed for fatty acid composition. Seeds were analyzedusing a 10-15 seed sample size. The results of the analysis are inTables 31 and 32. TABLE 31 Fatty acid composition of selfed F3 lines of97HEHOA [HE101 × (048 × 052)] Fatty Acid Composition (%) Sample No.C16:0 C18:0 C18:1 C18:2 C18:3 C20:0 C20:1 C22:0 C22:1 C24:0 C24:197HEIIOA-74 2.51 1.03 27.44 3.72 3.55 0.65 13.60 0.30 45.57 0.14 1.0397HEIIOA-01 3.66 1.40 47.79 6.47 2.97 0.62 19.23 0.25 16.11 0.14 0.7697HEIIOA-67 2.47 0.84 20.43 7.25 3.89 0.69 10.33 0.47 52.09 0.16 0.8697HEIIOA-59 2.81 1.08 27.01 7.88 2.82 0.69 16.15 0.32 39.68 0.15 0.8697HEHOA-88 3.16 1.21 44.85 9.21 2.31 0.49 16.30 0.21 21.00 0.12 0.5897HEHOA-33 2.53 0.79 21.90 9.52 3.55 0.53 11.51 0.31 47.59 0.13 1.0897HEHOA-5 2.93 1.01 23.67 10.26 2.00 0.63 14.34 0.38 42.98 0.15 1.06

[0175] TABLE 32 Fatty acid composition of selfed F3 lines of 97HEHOC[HE101 × (Q4275 × 663 − 40)] Fatty Acid Composition (%) Sample No. C16:0C18:0 C18:1 C18:2 C18:3 C20:0 C20:1 C22:0 C22:1 C24:0 C24:1 97HEHOC-2142.47 1.12 31.15 3.77 3.84 0.77 13.78 0.43 41.15 0.17 0.97 97HEHOC-2672.62 1.42 31.64 6.44 1.30 0.84 15.64 0.39 38.15 0.16 0.95 97HEHOC-2932.73 1.13 32.08 7.23 2.18 0.72 14.88 0.41 37.17 0.17 0.81 97HEHOC-2382.90 1.05 35.20 9.37 1.76 0.66 14.88 0.38 32.05 0.1 1.01 97HEHOC(2)-502.60 0.93 31.16 5.66 2.09 0.61 14.93 0.31 40.30 0.11 0.88 97HEHOC(2)-1563.19 1.71 46.56 3.05 1.59 0.94 16.41 0.40 24.67 0.19 0.83

[0176] To the extent not already indicated, it will be understood bythose of ordinary skill in the art that any one of the various specificembodiments herein described and illustrated may be further modified toincorporate features shown in other of the specific embodiments.

[0177] The foregoing detailed description has been provided for a betterunderstanding of the invention only and no unnecessary limitation shouldbe understood therefrom as some modifications will be apparent to thoseskilled in the art without deviating from the spirit and scope of theappended claims.

1 68 1 1155 DNA Brassica napus CDS (1)...(1152) Wild type Fad2 1 atg ggtgca ggt gga aga atg caa gtg tct cct ccc tcc aag aag tct 48 Met Gly AlaGly Gly Arg Met Gln Val Ser Pro Pro Ser Lys Lys Ser 1 5 10 15 gaa accgac acc atc aag cgc gta ccc tgc gag aca ccg ccc ttc act 96 Glu Thr AspThr Ile Lys Arg Val Pro Cys Glu Thr Pro Pro Phe Thr 20 25 30 gtc gga gaactc aag aaa gca atc cca ccg cac tgt ttc aaa cgc tcg 144 Val Gly Glu LeuLys Lys Ala Ile Pro Pro His Cys Phe Lys Arg Ser 35 40 45 atc cct cgc tctttc tcc tac ctc atc tgg gac atc atc ata gcc tcc 192 Ile Pro Arg Ser PheSer Tyr Leu Ile Trp Asp Ile Ile Ile Ala Ser 50 55 60 tgc ttc tac tac ntcgcc acc act tac ttc cct ctc ctc cct cac cct 240 Cys Phe Tyr Tyr Xaa AlaThr Thr Tyr Phe Pro Leu Leu Pro His Pro 65 70 75 80 ctc tcc tac ttc gcctgg cct ctc tac tgg gcc tgc caa ggg tgc gtc 288 Leu Ser Tyr Phe Ala TrpPro Leu Tyr Trp Ala Cys Gln Gly Cys Val 85 90 95 cta acc ggc gtc tgg gtcata gcc cac gaa tgc ggc cac cac gcc ttc 336 Leu Thr Gly Val Trp Val IleAla His Glu Cys Gly His His Ala Phe 100 105 110 agc gac tac cag tgg cttgac gac acc gtc ggt ctc atc ttc cac tcc 384 Ser Asp Tyr Gln Trp Leu AspAsp Thr Val Gly Leu Ile Phe His Ser 115 120 125 ttc ctc ctc gtc cct tacttc tcc tgg aag tac agt cat cgc agc cac 432 Phe Leu Leu Val Pro Tyr PheSer Trp Lys Tyr Ser His Arg Ser His 130 135 140 cat tcc aac act ggc tccctc gag aga gac gaa gtg ttt gtc ccc aag 480 His Ser Asn Thr Gly Ser LeuGlu Arg Asp Glu Val Phe Val Pro Lys 145 150 155 160 aag aag tca gac atcaag tgg tac ggc aag tac ctc aac aac cct ttg 528 Lys Lys Ser Asp Ile LysTrp Tyr Gly Lys Tyr Leu Asn Asn Pro Leu 165 170 175 gga cgc acc gtg atgtta acg gtt cag ttc act ctc ggc tgg ccg ttg 576 Gly Arg Thr Val Met LeuThr Val Gln Phe Thr Leu Gly Trp Pro Leu 180 185 190 tac tta gcc ttc aacgtc tcg gga aga cct tac gac ggc ggc ttc cgt 624 Tyr Leu Ala Phe Asn ValSer Gly Arg Pro Tyr Asp Gly Gly Phe Arg 195 200 205 tgc cat ttc cac cccaac gct ccc atc tac aac gac cgc gag cgt ctc 672 Cys His Phe His Pro AsnAla Pro Ile Tyr Asn Asp Arg Glu Arg Leu 210 215 220 cag ata tac atc tccgac gct ggc atc ctc gcc gtc tgc tac ggt ctc 720 Gln Ile Tyr Ile Ser AspAla Gly Ile Leu Ala Val Cys Tyr Gly Leu 225 230 235 240 ttc cgt tac gccgcc ggc cag gga gtg gcc tcg atg gtc tgc ttc tac 768 Phe Arg Tyr Ala AlaGly Gln Gly Val Ala Ser Met Val Cys Phe Tyr 245 250 255 gga gtc ccg cttctg att gtc aat ggt ttc ctc gtg ttg atc act tac 816 Gly Val Pro Leu LeuIle Val Asn Gly Phe Leu Val Leu Ile Thr Tyr 260 265 270 ttg cag cac acgcat cct tcc ctg cct cac tac gat tcg tcc gag tgg 864 Leu Gln His Thr HisPro Ser Leu Pro His Tyr Asp Ser Ser Glu Trp 275 280 285 gat tgg ttc agggga gct ttg gct acc gtt gac aga gac tac gga atc 912 Asp Trp Phe Arg GlyAla Leu Ala Thr Val Asp Arg Asp Tyr Gly Ile 290 295 300 ttg aac aag gtcttc cac aat att acc gac acg cac gtg gcc cat cat 960 Leu Asn Lys Val PheHis Asn Ile Thr Asp Thr His Val Ala His His 305 310 315 320 ccg ttc tccacg atg ccg cat tat cac gcg atg gaa gct acc aag gcg 1008 Pro Phe Ser ThrMet Pro His Tyr His Ala Met Glu Ala Thr Lys Ala 325 330 335 ata aag ccgata ctg gga gag tat tat cag ttc gat ggg acg ccg gtg 1056 Ile Lys Pro IleLeu Gly Glu Tyr Tyr Gln Phe Asp Gly Thr Pro Val 340 345 350 gtt aag gcgatg tgg agg gag gcg aag gag tgt atc tat gtg gaa ccg 1104 Val Lys Ala MetTrp Arg Glu Ala Lys Glu Cys Ile Tyr Val Glu Pro 355 360 365 gac agg caaggt gag aag aaa ggt gtg ttc tgg tac aac aat aag tta 1152 Asp Arg Gln GlyGlu Lys Lys Gly Val Phe Trp Tyr Asn Asn Lys Leu 370 375 380 tga 1155 2384 PRT Brassica napus Xaa = Phe, Leu, Ile, or Val 2 Met Gly Ala Gly GlyArg Met Gln Val Ser Pro Pro Ser Lys Lys Ser 1 5 10 15 Glu Thr Asp ThrIle Lys Arg Val Pro Cys Glu Thr Pro Pro Phe Thr 20 25 30 Val Gly Glu LeuLys Lys Ala Ile Pro Pro His Cys Phe Lys Arg Ser 35 40 45 Ile Pro Arg SerPhe Ser Tyr Leu Ile Trp Asp Ile Ile Ile Ala Ser 50 55 60 Cys Phe Tyr TyrXaa Ala Thr Thr Tyr Phe Pro Leu Leu Pro His Pro 65 70 75 80 Leu Ser TyrPhe Ala Trp Pro Leu Tyr Trp Ala Cys Gln Gly Cys Val 85 90 95 Leu Thr GlyVal Trp Val Ile Ala His Glu Cys Gly His His Ala Phe 100 105 110 Ser AspTyr Gln Trp Leu Asp Asp Thr Val Gly Leu Ile Phe His Ser 115 120 125 PheLeu Leu Val Pro Tyr Phe Ser Trp Lys Tyr Ser His Arg Ser His 130 135 140His Ser Asn Thr Gly Ser Leu Glu Arg Asp Glu Val Phe Val Pro Lys 145 150155 160 Lys Lys Ser Asp Ile Lys Trp Tyr Gly Lys Tyr Leu Asn Asn Pro Leu165 170 175 Gly Arg Thr Val Met Leu Thr Val Gln Phe Thr Leu Gly Trp ProLeu 180 185 190 Tyr Leu Ala Phe Asn Val Ser Gly Arg Pro Tyr Asp Gly GlyPhe Arg 195 200 205 Cys His Phe His Pro Asn Ala Pro Ile Tyr Asn Asp ArgGlu Arg Leu 210 215 220 Gln Ile Tyr Ile Ser Asp Ala Gly Ile Leu Ala ValCys Tyr Gly Leu 225 230 235 240 Phe Arg Tyr Ala Ala Gly Gln Gly Val AlaSer Met Val Cys Phe Tyr 245 250 255 Gly Val Pro Leu Leu Ile Val Asn GlyPhe Leu Val Leu Ile Thr Tyr 260 265 270 Leu Gln His Thr His Pro Ser LeuPro His Tyr Asp Ser Ser Glu Trp 275 280 285 Asp Trp Phe Arg Gly Ala LeuAla Thr Val Asp Arg Asp Tyr Gly Ile 290 295 300 Leu Asn Lys Val Phe HisAsn Ile Thr Asp Thr His Val Ala His His 305 310 315 320 Pro Phe Ser ThrMet Pro His Tyr His Ala Met Glu Ala Thr Lys Ala 325 330 335 Ile Lys ProIle Leu Gly Glu Tyr Tyr Gln Phe Asp Gly Thr Pro Val 340 345 350 Val LysAla Met Trp Arg Glu Ala Lys Glu Cys Ile Tyr Val Glu Pro 355 360 365 AspArg Gln Gly Glu Lys Lys Gly Val Phe Trp Tyr Asn Asn Lys Leu 370 375 3803 1155 DNA Brassica napus CDS (1)...(1152) g to a transversion mutationat nucleotide 316 3 atg ggt gca ggt gga aga atg caa gtg tct cct ccc tccaag aag tct 48 Met Gly Ala Gly Gly Arg Met Gln Val Ser Pro Pro Ser LysLys Ser 1 5 10 15 gaa acc gac acc atc aag cgc gta ccc tgc gag aca ccgccc ttc act 96 Glu Thr Asp Thr Ile Lys Arg Val Pro Cys Glu Thr Pro ProPhe Thr 20 25 30 gtc gga gaa ctc aag aaa gca atc cca ccg cac tgt ttc aaacgc tcg 144 Val Gly Glu Leu Lys Lys Ala Ile Pro Pro His Cys Phe Lys ArgSer 35 40 45 atc cct cgc tct ttc tcc tac ctc atc tgg gac atc atc ata gcctcc 192 Ile Pro Arg Ser Phe Ser Tyr Leu Ile Trp Asp Ile Ile Ile Ala Ser50 55 60 tgc ttc tac tac ntc gcc acc act tac ttc cct ctc ctc cct cac cct240 Cys Phe Tyr Tyr Xaa Ala Thr Thr Tyr Phe Pro Leu Leu Pro His Pro 6570 75 80 ctc tcc tac ttc gcc tgg cct ctc tac tgg gcc tgc caa ggg tgc gtc288 Leu Ser Tyr Phe Ala Trp Pro Leu Tyr Trp Ala Cys Gln Gly Cys Val 8590 95 cta acc ggc gtc tgg gtc ata gcc cac aag tgc ggc cac cac gcc ttc336 Leu Thr Gly Val Trp Val Ile Ala His Lys Cys Gly His His Ala Phe 100105 110 agc gac tac cag tgg ctt gac gac acc gtc ggt ctc atc ttc cac tcc384 Ser Asp Tyr Gln Trp Leu Asp Asp Thr Val Gly Leu Ile Phe His Ser 115120 125 ttc ctc ctc gtc cct tac ttc tcc tgg aag tac agt cat cgc agc cac432 Phe Leu Leu Val Pro Tyr Phe Ser Trp Lys Tyr Ser His Arg Ser His 130135 140 cat tcc aac act ggc tcc ctc gag aga gac gaa gtg ttt gtc ccc aag480 His Ser Asn Thr Gly Ser Leu Glu Arg Asp Glu Val Phe Val Pro Lys 145150 155 160 aag aag tca gac atc aag tgg tac ggc aag tac ctc aac aac cctttg 528 Lys Lys Ser Asp Ile Lys Trp Tyr Gly Lys Tyr Leu Asn Asn Pro Leu165 170 175 gga cgc acc gtg atg tta acg gtt cag ttc act ctc ggc tgg ccgttg 576 Gly Arg Thr Val Met Leu Thr Val Gln Phe Thr Leu Gly Trp Pro Leu180 185 190 tac tta gcc ttc aac gtc tcg gga aga cct tac gac ggc ggc ttccgt 624 Tyr Leu Ala Phe Asn Val Ser Gly Arg Pro Tyr Asp Gly Gly Phe Arg195 200 205 tgc cat ttc cac ccc aac gct ccc atc tac aac gac cgc gag cgtctc 672 Cys His Phe His Pro Asn Ala Pro Ile Tyr Asn Asp Arg Glu Arg Leu210 215 220 cag ata tac atc tcc gac gct ggc atc ctc gcc gtc tgc tac ggtctc 720 Gln Ile Tyr Ile Ser Asp Ala Gly Ile Leu Ala Val Cys Tyr Gly Leu225 230 235 240 ttc cgt tac gcc gcc ggc cag gga gtg gcc tcg atg gtc tgcttc tac 768 Phe Arg Tyr Ala Ala Gly Gln Gly Val Ala Ser Met Val Cys PheTyr 245 250 255 gga gtc ccg ctt ctg att gtc aat ggt ttc ctc gtg ttg atcact tac 816 Gly Val Pro Leu Leu Ile Val Asn Gly Phe Leu Val Leu Ile ThrTyr 260 265 270 ttg cag cac acg cat cct tcc ctg cct cac tac gat tcg tccgag tgg 864 Leu Gln His Thr His Pro Ser Leu Pro His Tyr Asp Ser Ser GluTrp 275 280 285 gat tgg ttc agg gga gct ttg gct acc gtt gac aga gac tacgga atc 912 Asp Trp Phe Arg Gly Ala Leu Ala Thr Val Asp Arg Asp Tyr GlyIle 290 295 300 ttg aac aag gtc ttc cac aat att acc gac acg cac gtg gcccat cat 960 Leu Asn Lys Val Phe His Asn Ile Thr Asp Thr His Val Ala HisHis 305 310 315 320 ccg ttc tcc acg atg ccg cat tat cac gcg atg gaa gctacc aag gcg 1008 Pro Phe Ser Thr Met Pro His Tyr His Ala Met Glu Ala ThrLys Ala 325 330 335 ata aag ccg ata ctg gga gag tat tat cag ttc gat gggacg ccg gtg 1056 Ile Lys Pro Ile Leu Gly Glu Tyr Tyr Gln Phe Asp Gly ThrPro Val 340 345 350 gtt aag gcg atg tgg agg gag gcg aag gag tgt atc tatgtg gaa ccg 1104 Val Lys Ala Met Trp Arg Glu Ala Lys Glu Cys Ile Tyr ValGlu Pro 355 360 365 gac agg caa ggt gag aag aaa ggt gtg ttc tgg tac aacaat aag tta 1152 Asp Arg Gln Gly Glu Lys Lys Gly Val Phe Trp Tyr Asn AsnLys Leu 370 375 380 tga 1155 4 384 PRT Brassica napus Xaa = Phe, Leu,Ile, or Val 4 Met Gly Ala Gly Gly Arg Met Gln Val Ser Pro Pro Ser LysLys Ser 1 5 10 15 Glu Thr Asp Thr Ile Lys Arg Val Pro Cys Glu Thr ProPro Phe Thr 20 25 30 Val Gly Glu Leu Lys Lys Ala Ile Pro Pro His Cys PheLys Arg Ser 35 40 45 Ile Pro Arg Ser Phe Ser Tyr Leu Ile Trp Asp Ile IleIle Ala Ser 50 55 60 Cys Phe Tyr Tyr Xaa Ala Thr Thr Tyr Phe Pro Leu LeuPro His Pro 65 70 75 80 Leu Ser Tyr Phe Ala Trp Pro Leu Tyr Trp Ala CysGln Gly Cys Val 85 90 95 Leu Thr Gly Val Trp Val Ile Ala His Lys Cys GlyHis His Ala Phe 100 105 110 Ser Asp Tyr Gln Trp Leu Asp Asp Thr Val GlyLeu Ile Phe His Ser 115 120 125 Phe Leu Leu Val Pro Tyr Phe Ser Trp LysTyr Ser His Arg Ser His 130 135 140 His Ser Asn Thr Gly Ser Leu Glu ArgAsp Glu Val Phe Val Pro Lys 145 150 155 160 Lys Lys Ser Asp Ile Lys TrpTyr Gly Lys Tyr Leu Asn Asn Pro Leu 165 170 175 Gly Arg Thr Val Met LeuThr Val Gln Phe Thr Leu Gly Trp Pro Leu 180 185 190 Tyr Leu Ala Phe AsnVal Ser Gly Arg Pro Tyr Asp Gly Gly Phe Arg 195 200 205 Cys His Phe HisPro Asn Ala Pro Ile Tyr Asn Asp Arg Glu Arg Leu 210 215 220 Gln Ile TyrIle Ser Asp Ala Gly Ile Leu Ala Val Cys Tyr Gly Leu 225 230 235 240 PheArg Tyr Ala Ala Gly Gln Gly Val Ala Ser Met Val Cys Phe Tyr 245 250 255Gly Val Pro Leu Leu Ile Val Asn Gly Phe Leu Val Leu Ile Thr Tyr 260 265270 Leu Gln His Thr His Pro Ser Leu Pro His Tyr Asp Ser Ser Glu Trp 275280 285 Asp Trp Phe Arg Gly Ala Leu Ala Thr Val Asp Arg Asp Tyr Gly Ile290 295 300 Leu Asn Lys Val Phe His Asn Ile Thr Asp Thr His Val Ala HisHis 305 310 315 320 Pro Phe Ser Thr Met Pro His Tyr His Ala Met Glu AlaThr Lys Ala 325 330 335 Ile Lys Pro Ile Leu Gly Glu Tyr Tyr Gln Phe AspGly Thr Pro Val 340 345 350 Val Lys Ala Met Trp Arg Glu Ala Lys Glu CysIle Tyr Val Glu Pro 355 360 365 Asp Arg Gln Gly Glu Lys Lys Gly Val PheTrp Tyr Asn Asn Lys Leu 370 375 380 5 1155 DNA Brassica napus CDS(1)...(1152) Wild type Fad2 5 atg ggt gca ggt gga aga atg caa gtg tctcct ccc tcc aaa aag tct 48 Met Gly Ala Gly Gly Arg Met Gln Val Ser ProPro Ser Lys Lys Ser 1 5 10 15 gaa acc gac aac atc aag cgc gta ccc tgcgag aca ccg ccc ttc act 96 Glu Thr Asp Asn Ile Lys Arg Val Pro Cys GluThr Pro Pro Phe Thr 20 25 30 gtc gga gaa ctc aag aaa gca atc cca ccg cactgt ttc aaa cgc tcg 144 Val Gly Glu Leu Lys Lys Ala Ile Pro Pro His CysPhe Lys Arg Ser 35 40 45 atc cct cgc tct ttc tcc tac ctc atc tgg gac atcatc ata gcc tcc 192 Ile Pro Arg Ser Phe Ser Tyr Leu Ile Trp Asp Ile IleIle Ala Ser 50 55 60 tgc ttc tac tac gtc gcc acc act tac ttc cct ctc ctccct cac cct 240 Cys Phe Tyr Tyr Val Ala Thr Thr Tyr Phe Pro Leu Leu ProHis Pro 65 70 75 80 ctc tcc tac ttc gcc tgg cct ctc tac tgg gcc tgc cagggc tgc gtc 288 Leu Ser Tyr Phe Ala Trp Pro Leu Tyr Trp Ala Cys Gln GlyCys Val 85 90 95 cta acc ggc gtc tgg gtc ata gcc cac gag tgc ggc cac cacgcc ttc 336 Leu Thr Gly Val Trp Val Ile Ala His Glu Cys Gly His His AlaPhe 100 105 110 agc gac tac cag tgg ctg gac gac acc gtc ggc ctc atc ttccac tcc 384 Ser Asp Tyr Gln Trp Leu Asp Asp Thr Val Gly Leu Ile Phe HisSer 115 120 125 ttc ctc ctc gtc cct tac ttc tcc tgg aag tac agt cat cgacgc cac 432 Phe Leu Leu Val Pro Tyr Phe Ser Trp Lys Tyr Ser His Arg ArgHis 130 135 140 cat tcc aac act ggc tcc ctc gag aga gac gaa gtg ttt gtcccc aag 480 His Ser Asn Thr Gly Ser Leu Glu Arg Asp Glu Val Phe Val ProLys 145 150 155 160 aag aag tca gac atc aag tgg tac ggc aag tac ctc aacaac cct ttg 528 Lys Lys Ser Asp Ile Lys Trp Tyr Gly Lys Tyr Leu Asn AsnPro Leu 165 170 175 gga cgc acc gtg atg tta acg gtt cag ttc act ctc ggctgg cct ttg 576 Gly Arg Thr Val Met Leu Thr Val Gln Phe Thr Leu Gly TrpPro Leu 180 185 190 tac tta gcc ttc aac gtc tcg ggg aga cct tac gac ggcggc ttc gct 624 Tyr Leu Ala Phe Asn Val Ser Gly Arg Pro Tyr Asp Gly GlyPhe Ala 195 200 205 tgc cat ttc cac ccc aac gct ccc atc tac aac gac cgcgag cgt ctc 672 Cys His Phe His Pro Asn Ala Pro Ile Tyr Asn Asp Arg GluArg Leu 210 215 220 cag ata tac atc tcc gac gct ggc atc ctc gcc gtc tgctac ggt ctc 720 Gln Ile Tyr Ile Ser Asp Ala Gly Ile Leu Ala Val Cys TyrGly Leu 225 230 235 240 tac cgc tac gct gct gtc caa gga gtt gcc tcg atggtc tgc ttc tac 768 Tyr Arg Tyr Ala Ala Val Gln Gly Val Ala Ser Met ValCys Phe Tyr 245 250 255 gga gtt ccg ctt ctg att gtc aat ggg ttc tta gttttg atc act tac 816 Gly Val Pro Leu Leu Ile Val Asn Gly Phe Leu Val LeuIle Thr Tyr 260 265 270 ttg cag cac acg cat cct tcc ctg cct cac tat gactcg tct gag tgg 864 Leu Gln His Thr His Pro Ser Leu Pro His Tyr Asp SerSer Glu Trp 275 280 285 gat tgg ttg agg gga gct ttg gcc acc gtt gac agagac tac gga atc 912 Asp Trp Leu Arg Gly Ala Leu Ala Thr Val Asp Arg AspTyr Gly Ile 290 295 300 ttg aac aag gtc ttc cac aat atc acg gac acg cacgtg gcg cat cac 960 Leu Asn Lys Val Phe His Asn Ile Thr Asp Thr His ValAla His His 305 310 315 320 ctg ttc tcg acc atg ccg cat tat cat gcg atggaa gct acg aag gcg 1008 Leu Phe Ser Thr Met Pro His Tyr His Ala Met GluAla Thr Lys Ala 325 330 335 ata aag ccg ata ctg gga gag tat tat cag ttgcat ggg acg ccg gtg 1056 Ile Lys Pro Ile Leu Gly Glu Tyr Tyr Gln Leu HisGly Thr Pro Val 340 345 350 gtt aag gcg atg tgg agg gag gcg aag gag tgtatc tat gtg gaa ccg 1104 Val Lys Ala Met Trp Arg Glu Ala Lys Glu Cys IleTyr Val Glu Pro 355 360 365 gac agg caa ggt gag aag aaa ggt gtg ttc tggtac aac aat aag tta 1152 Asp Arg Gln Gly Glu Lys Lys Gly Val Phe Trp TyrAsn Asn Lys Leu 370 375 380 tga 1155 6 384 PRT Brassica napus 6 Met GlyAla Gly Gly Arg Met Gln Val Ser Pro Pro Ser Lys Lys Ser 1 5 10 15 GluThr Asp Asn Ile Lys Arg Val Pro Cys Glu Thr Pro Pro Phe Thr 20 25 30 ValGly Glu Leu Lys Lys Ala Ile Pro Pro His Cys Phe Lys Arg Ser 35 40 45 IlePro Arg Ser Phe Ser Tyr Leu Ile Trp Asp Ile Ile Ile Ala Ser 50 55 60 CysPhe Tyr Tyr Val Ala Thr Thr Tyr Phe Pro Leu Leu Pro His Pro 65 70 75 80Leu Ser Tyr Phe Ala Trp Pro Leu Tyr Trp Ala Cys Gln Gly Cys Val 85 90 95Leu Thr Gly Val Trp Val Ile Ala His Glu Cys Gly His His Ala Phe 100 105110 Ser Asp Tyr Gln Trp Leu Asp Asp Thr Val Gly Leu Ile Phe His Ser 115120 125 Phe Leu Leu Val Pro Tyr Phe Ser Trp Lys Tyr Ser His Arg Arg His130 135 140 His Ser Asn Thr Gly Ser Leu Glu Arg Asp Glu Val Phe Val ProLys 145 150 155 160 Lys Lys Ser Asp Ile Lys Trp Tyr Gly Lys Tyr Leu AsnAsn Pro Leu 165 170 175 Gly Arg Thr Val Met Leu Thr Val Gln Phe Thr LeuGly Trp Pro Leu 180 185 190 Tyr Leu Ala Phe Asn Val Ser Gly Arg Pro TyrAsp Gly Gly Phe Ala 195 200 205 Cys His Phe His Pro Asn Ala Pro Ile TyrAsn Asp Arg Glu Arg Leu 210 215 220 Gln Ile Tyr Ile Ser Asp Ala Gly IleLeu Ala Val Cys Tyr Gly Leu 225 230 235 240 Tyr Arg Tyr Ala Ala Val GlnGly Val Ala Ser Met Val Cys Phe Tyr 245 250 255 Gly Val Pro Leu Leu IleVal Asn Gly Phe Leu Val Leu Ile Thr Tyr 260 265 270 Leu Gln His Thr HisPro Ser Leu Pro His Tyr Asp Ser Ser Glu Trp 275 280 285 Asp Trp Leu ArgGly Ala Leu Ala Thr Val Asp Arg Asp Tyr Gly Ile 290 295 300 Leu Asn LysVal Phe His Asn Ile Thr Asp Thr His Val Ala His His 305 310 315 320 LeuPhe Ser Thr Met Pro His Tyr His Ala Met Glu Ala Thr Lys Ala 325 330 335Ile Lys Pro Ile Leu Gly Glu Tyr Tyr Gln Leu His Gly Thr Pro Val 340 345350 Val Lys Ala Met Trp Arg Glu Ala Lys Glu Cys Ile Tyr Val Glu Pro 355360 365 Asp Arg Gln Gly Glu Lys Lys Gly Val Phe Trp Tyr Asn Asn Lys Leu370 375 380 7 1155 DNA Brassica napus CDS (1)...(1152) T to Atransversion mutation at nucleotide 515 7 atg ggt gca ggt gga aga atgcaa gtg tct cct ccc tcc aaa aag tct 48 Met Gly Ala Gly Gly Arg Met GlnVal Ser Pro Pro Ser Lys Lys Ser 1 5 10 15 gaa acc gac aac atc aag cgcgta ccc tgc gag aca ccg ccc ttc act 96 Glu Thr Asp Asn Ile Lys Arg ValPro Cys Glu Thr Pro Pro Phe Thr 20 25 30 gtc gga gaa ctc aag aaa gca atccca ccg cac tgt ttc aaa cgc tcg 144 Val Gly Glu Leu Lys Lys Ala Ile ProPro His Cys Phe Lys Arg Ser 35 40 45 atc cct cgc tct ttc tcc tac ctc atctgg gac atc atc ata gcc tcc 192 Ile Pro Arg Ser Phe Ser Tyr Leu Ile TrpAsp Ile Ile Ile Ala Ser 50 55 60 tgc ttc tac tac gtc gcc acc act tac ttccct ctc ctc cct cac cct 240 Cys Phe Tyr Tyr Val Ala Thr Thr Tyr Phe ProLeu Leu Pro His Pro 65 70 75 80 ctc tcc tac ttc gcc tgg cct ctc tac tgggcc tgc cag ggc tgc gtc 288 Leu Ser Tyr Phe Ala Trp Pro Leu Tyr Trp AlaCys Gln Gly Cys Val 85 90 95 cta acc ggc gtc tgg gtc ata gcc cac gag tgcggc cac cac gcc ttc 336 Leu Thr Gly Val Trp Val Ile Ala His Glu Cys GlyHis His Ala Phe 100 105 110 agc gac tac cag tgg ctg gac gac acc gtc ggcctc atc ttc cac tcc 384 Ser Asp Tyr Gln Trp Leu Asp Asp Thr Val Gly LeuIle Phe His Ser 115 120 125 ttc ctc ctc gtc cct tac ttc tcc tgg aag tacagt cat cga cgc cac 432 Phe Leu Leu Val Pro Tyr Phe Ser Trp Lys Tyr SerHis Arg Arg His 130 135 140 cat tcc aac act ggc tcc ctc gag aga gac gaagtg ttt gtc ccc aag 480 His Ser Asn Thr Gly Ser Leu Glu Arg Asp Glu ValPhe Val Pro Lys 145 150 155 160 aag aag tca gac atc aag tgg tac ggc aagtac cac aac aac cct ttg 528 Lys Lys Ser Asp Ile Lys Trp Tyr Gly Lys TyrHis Asn Asn Pro Leu 165 170 175 gga cgc acc gtg atg tta acg gtt cag ttcact ctc ggc tgg cct ttg 576 Gly Arg Thr Val Met Leu Thr Val Gln Phe ThrLeu Gly Trp Pro Leu 180 185 190 tac tta gcc ttc aac gtc tcg ggg aga ccttac gac ggc ggc ttc gct 624 Tyr Leu Ala Phe Asn Val Ser Gly Arg Pro TyrAsp Gly Gly Phe Ala 195 200 205 tgc cat ttc cac ccc aac gct ccc atc tacaac gac cgc gag cgt ctc 672 Cys His Phe His Pro Asn Ala Pro Ile Tyr AsnAsp Arg Glu Arg Leu 210 215 220 cag ata tac atc tcc gac gct ggc atc ctcgcc gtc tgc tac ggt ctc 720 Gln Ile Tyr Ile Ser Asp Ala Gly Ile Leu AlaVal Cys Tyr Gly Leu 225 230 235 240 tac cgc tac gct gct gtc caa gga gttgcc tcg atg gtc tgc ttc tac 768 Tyr Arg Tyr Ala Ala Val Gln Gly Val AlaSer Met Val Cys Phe Tyr 245 250 255 gga gtt ccg ctt ctg att gtc aat gggttc tta gtt ttg atc act tac 816 Gly Val Pro Leu Leu Ile Val Asn Gly PheLeu Val Leu Ile Thr Tyr 260 265 270 ttg cag cac acg cat cct tcc ctg cctcac tat gac tcg tct gag tgg 864 Leu Gln His Thr His Pro Ser Leu Pro HisTyr Asp Ser Ser Glu Trp 275 280 285 gat tgg ttg agg gga gct ttg gcc accgtt gac aga gac tac gga atc 912 Asp Trp Leu Arg Gly Ala Leu Ala Thr ValAsp Arg Asp Tyr Gly Ile 290 295 300 ttg aac aag gtc ttc cac aat atc acggac acg cac gtg gcg cat cac 960 Leu Asn Lys Val Phe His Asn Ile Thr AspThr His Val Ala His His 305 310 315 320 ctg ttc tcg acc atg ccg cat tatcat gcg atg gaa gct acg aag gcg 1008 Leu Phe Ser Thr Met Pro His Tyr HisAla Met Glu Ala Thr Lys Ala 325 330 335 ata aag ccg ata ctg gga gag tattat cag ttg cat ggg acg ccg gtg 1056 Ile Lys Pro Ile Leu Gly Glu Tyr TyrGln Leu His Gly Thr Pro Val 340 345 350 gtt aag gcg atg tgg agg gag gcgaag gag tgt atc tat gtg gaa ccg 1104 Val Lys Ala Met Trp Arg Glu Ala LysGlu Cys Ile Tyr Val Glu Pro 355 360 365 gac agg caa ggt gag aag aaa ggtgtg ttc tgg tac aac aat aag tta 1152 Asp Arg Gln Gly Glu Lys Lys Gly ValPhe Trp Tyr Asn Asn Lys Leu 370 375 380 tga 1155 8 384 PRT Brassicanapus 8 Met Gly Ala Gly Gly Arg Met Gln Val Ser Pro Pro Ser Lys Lys Ser1 5 10 15 Glu Thr Asp Asn Ile Lys Arg Val Pro Cys Glu Thr Pro Pro PheThr 20 25 30 Val Gly Glu Leu Lys Lys Ala Ile Pro Pro His Cys Phe Lys ArgSer 35 40 45 Ile Pro Arg Ser Phe Ser Tyr Leu Ile Trp Asp Ile Ile Ile AlaSer 50 55 60 Cys Phe Tyr Tyr Val Ala Thr Thr Tyr Phe Pro Leu Leu Pro HisPro 65 70 75 80 Leu Ser Tyr Phe Ala Trp Pro Leu Tyr Trp Ala Cys Gln GlyCys Val 85 90 95 Leu Thr Gly Val Trp Val Ile Ala His Glu Cys Gly His HisAla Phe 100 105 110 Ser Asp Tyr Gln Trp Leu Asp Asp Thr Val Gly Leu IlePhe His Ser 115 120 125 Phe Leu Leu Val Pro Tyr Phe Ser Trp Lys Tyr SerHis Arg Arg His 130 135 140 His Ser Asn Thr Gly Ser Leu Glu Arg Asp GluVal Phe Val Pro Lys 145 150 155 160 Lys Lys Ser Asp Ile Lys Trp Tyr GlyLys Tyr His Asn Asn Pro Leu 165 170 175 Gly Arg Thr Val Met Leu Thr ValGln Phe Thr Leu Gly Trp Pro Leu 180 185 190 Tyr Leu Ala Phe Asn Val SerGly Arg Pro Tyr Asp Gly Gly Phe Ala 195 200 205 Cys His Phe His Pro AsnAla Pro Ile Tyr Asn Asp Arg Glu Arg Leu 210 215 220 Gln Ile Tyr Ile SerAsp Ala Gly Ile Leu Ala Val Cys Tyr Gly Leu 225 230 235 240 Tyr Arg TyrAla Ala Val Gln Gly Val Ala Ser Met Val Cys Phe Tyr 245 250 255 Gly ValPro Leu Leu Ile Val Asn Gly Phe Leu Val Leu Ile Thr Tyr 260 265 270 LeuGln His Thr His Pro Ser Leu Pro His Tyr Asp Ser Ser Glu Trp 275 280 285Asp Trp Leu Arg Gly Ala Leu Ala Thr Val Asp Arg Asp Tyr Gly Ile 290 295300 Leu Asn Lys Val Phe His Asn Ile Thr Asp Thr His Val Ala His His 305310 315 320 Leu Phe Ser Thr Met Pro His Tyr His Ala Met Glu Ala Thr LysAla 325 330 335 Ile Lys Pro Ile Leu Gly Glu Tyr Tyr Gln Leu His Gly ThrPro Val 340 345 350 Val Lys Ala Met Trp Arg Glu Ala Lys Glu Cys Ile TyrVal Glu Pro 355 360 365 Asp Arg Gln Gly Glu Lys Lys Gly Val Phe Trp TyrAsn Asn Lys Leu 370 375 380 9 1155 DNA Brassica napus CDS (1)...(1152) 9atg ggt gca ggt gga aga atg caa gtg tct cct ccc tcc aaa aag tct 48 MetGly Ala Gly Gly Arg Met Gln Val Ser Pro Pro Ser Lys Lys Ser 1 5 10 15gaa acc gac aac atc aag cgc gta ccc tgc gag aca ccg ccc ttc act 96 GluThr Asp Asn Ile Lys Arg Val Pro Cys Glu Thr Pro Pro Phe Thr 20 25 30 gtcgga gaa ctc aag aaa gca atc cca ccg cac tgt ttc aaa cgc tcg 144 Val GlyGlu Leu Lys Lys Ala Ile Pro Pro His Cys Phe Lys Arg Ser 35 40 45 atc cctcgc tct ttc tcc tac ctc atc tgg gac atc atc ata gcc tcc 192 Ile Pro ArgSer Phe Ser Tyr Leu Ile Trp Asp Ile Ile Ile Ala Ser 50 55 60 tgc ttc tactac gtc gcc acc act tac ttc cct ctc ctc cct cac cct 240 Cys Phe Tyr TyrVal Ala Thr Thr Tyr Phe Pro Leu Leu Pro His Pro 65 70 75 80 ctc tcc tacttc gcc tgg cct ctc tac tgg gcc tgc cag ggc tgc gtc 288 Leu Ser Tyr PheAla Trp Pro Leu Tyr Trp Ala Cys Gln Gly Cys Val 85 90 95 cta acc ggc gtctgg gtc ata gcc cac gag tgc ggc cac cac gcc ttc 336 Leu Thr Gly Val TrpVal Ile Ala His Glu Cys Gly His His Ala Phe 100 105 110 agc gac tac cagtgg ctg gac gac acc gtc ggc ctc atc ttc cac tcc 384 Ser Asp Tyr Gln TrpLeu Asp Asp Thr Val Gly Leu Ile Phe His Ser 115 120 125 ttc ctc ctc gtccct tac ttc tcc tgg aag tac agt cat cga cgc cac 432 Phe Leu Leu Val ProTyr Phe Ser Trp Lys Tyr Ser His Arg Arg His 130 135 140 cat tcc aac actggc tcc ctc gag aga gac gaa gtg ttt gtc ccc aag 480 His Ser Asn Thr GlySer Leu Glu Arg Asp Glu Val Phe Val Pro Lys 145 150 155 160 aag aag tcagac atc aag tgg tac ggc aag tac ctc aac aac cct ttg 528 Lys Lys Ser AspIle Lys Trp Tyr Gly Lys Tyr Leu Asn Asn Pro Leu 165 170 175 gga cgc accgtg atg tta acg gtt cag ttc act ctc ggc tgg cct ttg 576 Gly Arg Thr ValMet Leu Thr Val Gln Phe Thr Leu Gly Trp Pro Leu 180 185 190 tac tta gccttc aac gtc tcg ggg aga cct tac gac ggc ggc ttc gct 624 Tyr Leu Ala PheAsn Val Ser Gly Arg Pro Tyr Asp Gly Gly Phe Ala 195 200 205 tgc cat ttccac ccc aac gct ccc atc tac aac gac cgt gag cgt ctc 672 Cys His Phe HisPro Asn Ala Pro Ile Tyr Asn Asp Arg Glu Arg Leu 210 215 220 cag ata tacatc tcc gac gct ggc atc ctc gcc gtc tgc tac ggt ctc 720 Gln Ile Tyr IleSer Asp Ala Gly Ile Leu Ala Val Cys Tyr Gly Leu 225 230 235 240 tac cgctac gct gct gtc caa gga gtt gcc tcg atg gtc tgc ttc tac 768 Tyr Arg TyrAla Ala Val Gln Gly Val Ala Ser Met Val Cys Phe Tyr 245 250 255 gga gttcct ctt ctg att gtc aac ggg ttc tta gtt ttg atc act tac 816 Gly Val ProLeu Leu Ile Val Asn Gly Phe Leu Val Leu Ile Thr Tyr 260 265 270 ttg cagcac acg cat cct tcc ctg cct cac tat gac tcg tct gag tgg 864 Leu Gln HisThr His Pro Ser Leu Pro His Tyr Asp Ser Ser Glu Trp 275 280 285 gat tggttg agg gga gct ttg gcc acc gtt gac aga gac tac gga atc 912 Asp Trp LeuArg Gly Ala Leu Ala Thr Val Asp Arg Asp Tyr Gly Ile 290 295 300 ttg aacaag gtc ttc cac aat atc acg gac acg cac gtg gcg cat cac 960 Leu Asn LysVal Phe His Asn Ile Thr Asp Thr His Val Ala His His 305 310 315 320 ctgttc tcg acc atg ccg cat tat cat gcg atg gaa gct acg aag gcg 1008 Leu PheSer Thr Met Pro His Tyr His Ala Met Glu Ala Thr Lys Ala 325 330 335 ataaag ccg ata ctg gga gag tat tat cag ttc gat ggg acg ccg gtg 1056 Ile LysPro Ile Leu Gly Glu Tyr Tyr Gln Phe Asp Gly Thr Pro Val 340 345 350 gttaag gcg atg tgg agg gag gcg aag gag tgt atc tat gtg gaa ccg 1104 Val LysAla Met Trp Arg Glu Ala Lys Glu Cys Ile Tyr Val Glu Pro 355 360 365 gacagg caa ggt gag aag aaa ggt gtg ttc tgg tac aac aat aag tta 1152 Asp ArgGln Gly Glu Lys Lys Gly Val Phe Trp Tyr Asn Asn Lys Leu 370 375 380 tga1155 10 384 PRT Brassica napus 10 Met Gly Ala Gly Gly Arg Met Gln ValSer Pro Pro Ser Lys Lys Ser 1 5 10 15 Glu Thr Asp Asn Ile Lys Arg ValPro Cys Glu Thr Pro Pro Phe Thr 20 25 30 Val Gly Glu Leu Lys Lys Ala IlePro Pro His Cys Phe Lys Arg Ser 35 40 45 Ile Pro Arg Ser Phe Ser Tyr LeuIle Trp Asp Ile Ile Ile Ala Ser 50 55 60 Cys Phe Tyr Tyr Val Ala Thr ThrTyr Phe Pro Leu Leu Pro His Pro 65 70 75 80 Leu Ser Tyr Phe Ala Trp ProLeu Tyr Trp Ala Cys Gln Gly Cys Val 85 90 95 Leu Thr Gly Val Trp Val IleAla His Glu Cys Gly His His Ala Phe 100 105 110 Ser Asp Tyr Gln Trp LeuAsp Asp Thr Val Gly Leu Ile Phe His Ser 115 120 125 Phe Leu Leu Val ProTyr Phe Ser Trp Lys Tyr Ser His Arg Arg His 130 135 140 His Ser Asn ThrGly Ser Leu Glu Arg Asp Glu Val Phe Val Pro Lys 145 150 155 160 Lys LysSer Asp Ile Lys Trp Tyr Gly Lys Tyr Leu Asn Asn Pro Leu 165 170 175 GlyArg Thr Val Met Leu Thr Val Gln Phe Thr Leu Gly Trp Pro Leu 180 185 190Tyr Leu Ala Phe Asn Val Ser Gly Arg Pro Tyr Asp Gly Gly Phe Ala 195 200205 Cys His Phe His Pro Asn Ala Pro Ile Tyr Asn Asp Arg Glu Arg Leu 210215 220 Gln Ile Tyr Ile Ser Asp Ala Gly Ile Leu Ala Val Cys Tyr Gly Leu225 230 235 240 Tyr Arg Tyr Ala Ala Val Gln Gly Val Ala Ser Met Val CysPhe Tyr 245 250 255 Gly Val Pro Leu Leu Ile Val Asn Gly Phe Leu Val LeuIle Thr Tyr 260 265 270 Leu Gln His Thr His Pro Ser Leu Pro His Tyr AspSer Ser Glu Trp 275 280 285 Asp Trp Leu Arg Gly Ala Leu Ala Thr Val AspArg Asp Tyr Gly Ile 290 295 300 Leu Asn Lys Val Phe His Asn Ile Thr AspThr His Val Ala His His 305 310 315 320 Leu Phe Ser Thr Met Pro His TyrHis Ala Met Glu Ala Thr Lys Ala 325 330 335 Ile Lys Pro Ile Leu Gly GluTyr Tyr Gln Phe Asp Gly Thr Pro Val 340 345 350 Val Lys Ala Met Trp ArgGlu Ala Lys Glu Cys Ile Tyr Val Glu Pro 355 360 365 Asp Arg Gln Gly GluLys Lys Gly Val Phe Trp Tyr Asn Asn Lys Leu 370 375 380 11 1155 DNABrassica napus CDS (1)...(1152) 11 atg ggt gca ggt gga aga atg caa gtgtct cct ccc tcc aaa aag tct 48 Met Gly Ala Gly Gly Arg Met Gln Val SerPro Pro Ser Lys Lys Ser 1 5 10 15 gaa acc gac aac atc aag cgc gta ccctgc gag aca ccg ccc ttc act 96 Glu Thr Asp Asn Ile Lys Arg Val Pro CysGlu Thr Pro Pro Phe Thr 20 25 30 gtc gga gaa ctc aag aaa gca atc cca ccgcac tgt ttc aaa cgc tcg 144 Val Gly Glu Leu Lys Lys Ala Ile Pro Pro HisCys Phe Lys Arg Ser 35 40 45 atc cct cgc tct ttc tcc tac ctc atc tgg gacatc atc ata gcc tcc 192 Ile Pro Arg Ser Phe Ser Tyr Leu Ile Trp Asp IleIle Ile Ala Ser 50 55 60 tgc ttc tac tac gtc gcc acc act tac ttc cct ctcctc cct cac cct 240 Cys Phe Tyr Tyr Val Ala Thr Thr Tyr Phe Pro Leu LeuPro His Pro 65 70 75 80 ctc tcc tac ttc gcc tgg cct ctc tac tgg gcc tgccag ggc tgc gtc 288 Leu Ser Tyr Phe Ala Trp Pro Leu Tyr Trp Ala Cys GlnGly Cys Val 85 90 95 cta acc ggc gtc tgg gtc ata gcc cac aag tgc ggc caccac gcc ttc 336 Leu Thr Gly Val Trp Val Ile Ala His Lys Cys Gly His HisAla Phe 100 105 110 agc gac tac cag tgg ctg gac gac acc gtc ggc ctc atcttc cac tcc 384 Ser Asp Tyr Gln Trp Leu Asp Asp Thr Val Gly Leu Ile PheHis Ser 115 120 125 ttc ctc ctc gtc cct tac ttc tcc tgg aag tac agt catcga cgc cac 432 Phe Leu Leu Val Pro Tyr Phe Ser Trp Lys Tyr Ser His ArgArg His 130 135 140 cat tcc aac act ggc tcc ctc gag aga gac gaa gtg tttgtc ccc aag 480 His Ser Asn Thr Gly Ser Leu Glu Arg Asp Glu Val Phe ValPro Lys 145 150 155 160 aag aag tca gac atc aag tgg tac ggc aag tac ctcaac aac cct ttg 528 Lys Lys Ser Asp Ile Lys Trp Tyr Gly Lys Tyr Leu AsnAsn Pro Leu 165 170 175 gga cgc acc gtg atg tta acg gtt cag ttc act ctcggc tgg cct ttg 576 Gly Arg Thr Val Met Leu Thr Val Gln Phe Thr Leu GlyTrp Pro Leu 180 185 190 tac tta gcc ttc aac gtc tcg ggg aga cct tac gacggc ggc ttc gct 624 Tyr Leu Ala Phe Asn Val Ser Gly Arg Pro Tyr Asp GlyGly Phe Ala 195 200 205 tgc cat ttc cac ccc aac gct ccc atc tac aac gaccgt gag cgt ctc 672 Cys His Phe His Pro Asn Ala Pro Ile Tyr Asn Asp ArgGlu Arg Leu 210 215 220 cag ata tac atc tcc gac gct ggc atc ctc gcc gtctgc tac ggt ctc 720 Gln Ile Tyr Ile Ser Asp Ala Gly Ile Leu Ala Val CysTyr Gly Leu 225 230 235 240 tac cgc tac gct gct gtc caa gga gtt gcc tcgatg gtc tgc ttc tac 768 Tyr Arg Tyr Ala Ala Val Gln Gly Val Ala Ser MetVal Cys Phe Tyr 245 250 255 gga gtt cct ctt ctg att gtc aac ggg ttc ttagtt ttg atc act tac 816 Gly Val Pro Leu Leu Ile Val Asn Gly Phe Leu ValLeu Ile Thr Tyr 260 265 270 ttg cag cac acg cat cct tcc ctg cct cac tatgac tcg tct gag tgg 864 Leu Gln His Thr His Pro Ser Leu Pro His Tyr AspSer Ser Glu Trp 275 280 285 gat tgg ttg agg gga gct ttg gcc acc gtt gacaga gac tac gga atc 912 Asp Trp Leu Arg Gly Ala Leu Ala Thr Val Asp ArgAsp Tyr Gly Ile 290 295 300 ttg aac aag gtc ttc cac aat atc acg gac acgcac gtg gcg cat cac 960 Leu Asn Lys Val Phe His Asn Ile Thr Asp Thr HisVal Ala His His 305 310 315 320 ctg ttc tcg acc atg ccg cat tat cat gcgatg gaa gct acg aag gcg 1008 Leu Phe Ser Thr Met Pro His Tyr His Ala MetGlu Ala Thr Lys Ala 325 330 335 ata aag ccg ata ctg gga gag tat tat cagttc gat ggg acg ccg gtg 1056 Ile Lys Pro Ile Leu Gly Glu Tyr Tyr Gln PheAsp Gly Thr Pro Val 340 345 350 gtt aag gcg atg tgg agg gag gcg aag gagtgt atc tat gtg gaa ccg 1104 Val Lys Ala Met Trp Arg Glu Ala Lys Glu CysIle Tyr Val Glu Pro 355 360 365 gac agg caa ggt gag aag aaa ggt gtg ttctgg tac aac aat aag tta 1152 Asp Arg Gln Gly Glu Lys Lys Gly Val Phe TrpTyr Asn Asn Lys Leu 370 375 380 tga 1155 12 384 PRT Brassica napus 12Met Gly Ala Gly Gly Arg Met Gln Val Ser Pro Pro Ser Lys Lys Ser 1 5 1015 Glu Thr Asp Asn Ile Lys Arg Val Pro Cys Glu Thr Pro Pro Phe Thr 20 2530 Val Gly Glu Leu Lys Lys Ala Ile Pro Pro His Cys Phe Lys Arg Ser 35 4045 Ile Pro Arg Ser Phe Ser Tyr Leu Ile Trp Asp Ile Ile Ile Ala Ser 50 5560 Cys Phe Tyr Tyr Val Ala Thr Thr Tyr Phe Pro Leu Leu Pro His Pro 65 7075 80 Leu Ser Tyr Phe Ala Trp Pro Leu Tyr Trp Ala Cys Gln Gly Cys Val 8590 95 Leu Thr Gly Val Trp Val Ile Ala His Lys Cys Gly His His Ala Phe100 105 110 Ser Asp Tyr Gln Trp Leu Asp Asp Thr Val Gly Leu Ile Phe HisSer 115 120 125 Phe Leu Leu Val Pro Tyr Phe Ser Trp Lys Tyr Ser His ArgArg His 130 135 140 His Ser Asn Thr Gly Ser Leu Glu Arg Asp Glu Val PheVal Pro Lys 145 150 155 160 Lys Lys Ser Asp Ile Lys Trp Tyr Gly Lys TyrLeu Asn Asn Pro Leu 165 170 175 Gly Arg Thr Val Met Leu Thr Val Gln PheThr Leu Gly Trp Pro Leu 180 185 190 Tyr Leu Ala Phe Asn Val Ser Gly ArgPro Tyr Asp Gly Gly Phe Ala 195 200 205 Cys His Phe His Pro Asn Ala ProIle Tyr Asn Asp Arg Glu Arg Leu 210 215 220 Gln Ile Tyr Ile Ser Asp AlaGly Ile Leu Ala Val Cys Tyr Gly Leu 225 230 235 240 Tyr Arg Tyr Ala AlaVal Gln Gly Val Ala Ser Met Val Cys Phe Tyr 245 250 255 Gly Val Pro LeuLeu Ile Val Asn Gly Phe Leu Val Leu Ile Thr Tyr 260 265 270 Leu Gln HisThr His Pro Ser Leu Pro His Tyr Asp Ser Ser Glu Trp 275 280 285 Asp TrpLeu Arg Gly Ala Leu Ala Thr Val Asp Arg Asp Tyr Gly Ile 290 295 300 LeuAsn Lys Val Phe His Asn Ile Thr Asp Thr His Val Ala His His 305 310 315320 Leu Phe Ser Thr Met Pro His Tyr His Ala Met Glu Ala Thr Lys Ala 325330 335 Ile Lys Pro Ile Leu Gly Glu Tyr Tyr Gln Phe Asp Gly Thr Pro Val340 345 350 Val Lys Ala Met Trp Arg Glu Ala Lys Glu Cys Ile Tyr Val GluPro 355 360 365 Asp Arg Gln Gly Glu Lys Lys Gly Val Phe Trp Tyr Asn AsnLys Leu 370 375 380 13 1155 DNA Brassica napus CDS (1)...(1152) 13 atgggt gca ggt gga aga atg caa gtg tct cct ccc tcc aag aag tct 48 Met GlyAla Gly Gly Arg Met Gln Val Ser Pro Pro Ser Lys Lys Ser 1 5 10 15 gaaacc gac acc atc aag cgc gta ccc tgc gag aca ccg ccc ttc act 96 Glu ThrAsp Thr Ile Lys Arg Val Pro Cys Glu Thr Pro Pro Phe Thr 20 25 30 gtc ggagaa ctc aag aaa gca atc cca ccg cac tgt ttc aaa cgc tcg 144 Val Gly GluLeu Lys Lys Ala Ile Pro Pro His Cys Phe Lys Arg Ser 35 40 45 atc cct cgctct ttc tcc tac ctc atc tgg gac atc atc ata gcc tcc 192 Ile Pro Arg SerPhe Ser Tyr Leu Ile Trp Asp Ile Ile Ile Ala Ser 50 55 60 tgc ttc tac tacgtc gcc acc act tac ttc cct ctc ctc cct cac cct 240 Cys Phe Tyr Tyr ValAla Thr Thr Tyr Phe Pro Leu Leu Pro His Pro 65 70 75 80 ctc tcc tac ttcgcc tgg cct ctc tac tgg gcc tgc caa ggg tgc gtc 288 Leu Ser Tyr Phe AlaTrp Pro Leu Tyr Trp Ala Cys Gln Gly Cys Val 85 90 95 cta acc ggc gtc tgggtc ata gcc cac gag tgc ggc cac cac gcc ttc 336 Leu Thr Gly Val Trp ValIle Ala His Glu Cys Gly His His Ala Phe 100 105 110 agc gac tac cag tggctt gac gac acc gtc ggt ctc atc ttc cac tcc 384 Ser Asp Tyr Gln Trp LeuAsp Asp Thr Val Gly Leu Ile Phe His Ser 115 120 125 ttc ctc ctc gtc ccttac ttc tcc tgg aag tac agt cat cga cgc cac 432 Phe Leu Leu Val Pro TyrPhe Ser Trp Lys Tyr Ser His Arg Arg His 130 135 140 cat tcc aac act ggctcc ctc gag aga gac gaa gtg ttt gtc ccc aag 480 His Ser Asn Thr Gly SerLeu Glu Arg Asp Glu Val Phe Val Pro Lys 145 150 155 160 aag aag tca gacatc aag tgg tac ggc aag tac ctc aac aac cct ttg 528 Lys Lys Ser Asp IleLys Trp Tyr Gly Lys Tyr Leu Asn Asn Pro Leu 165 170 175 gga cgc acc gtgatg tta acg gtt cag ttc act ctc ggc tgg ccg ttg 576 Gly Arg Thr Val MetLeu Thr Val Gln Phe Thr Leu Gly Trp Pro Leu 180 185 190 tac tta gcc ttcaac gtc tcg gga aga cct tac gac ggc ggc ttc gct 624 Tyr Leu Ala Phe AsnVal Ser Gly Arg Pro Tyr Asp Gly Gly Phe Ala 195 200 205 tgc cat ttc cacccc aac gct ccc atc tac aac gac cgc gag cgt ctc 672 Cys His Phe His ProAsn Ala Pro Ile Tyr Asn Asp Arg Glu Arg Leu 210 215 220 cag ata tac atctcc gac gct ggc atc ctc gcc gtc tgc tac ggt ctc 720 Gln Ile Tyr Ile SerAsp Ala Gly Ile Leu Ala Val Cys Tyr Gly Leu 225 230 235 240 ttc cgt tacgcc gcc gcg cag gga gtg gcc tcg atg gtc tgc ttc tac 768 Phe Arg Tyr AlaAla Ala Gln Gly Val Ala Ser Met Val Cys Phe Tyr 245 250 255 gga gtc ccgctt ctg att gtc aat ggt ttc ctc gtg ttg atc act tac 816 Gly Val Pro LeuLeu Ile Val Asn Gly Phe Leu Val Leu Ile Thr Tyr 260 265 270 ttg cag cacacg cat cct tcc ctg cct cac tac gat tcg tcc gag tgg 864 Leu Gln His ThrHis Pro Ser Leu Pro His Tyr Asp Ser Ser Glu Trp 275 280 285 gat tgg ttgagg gga gct ttg gct acc gtt gac aga gac tac gga atc 912 Asp Trp Leu ArgGly Ala Leu Ala Thr Val Asp Arg Asp Tyr Gly Ile 290 295 300 ttg aac aaggtc ttc cac aat att acc gac acg cac gtg gcg cat cat 960 Leu Asn Lys ValPhe His Asn Ile Thr Asp Thr His Val Ala His His 305 310 315 320 ctg ttctcc acg atg ccg cat tat cac gcg atg gaa gct acc aag gcg 1008 Leu Phe SerThr Met Pro His Tyr His Ala Met Glu Ala Thr Lys Ala 325 330 335 ata aagccg ata ctg gga gag tat tat cag ttc gat ggg acg ccg gtg 1056 Ile Lys ProIle Leu Gly Glu Tyr Tyr Gln Phe Asp Gly Thr Pro Val 340 345 350 gtt aaggcg atg tgg agg gag gcg aag gag tgt atc tat gtg gaa ccg 1104 Val Lys AlaMet Trp Arg Glu Ala Lys Glu Cys Ile Tyr Val Glu Pro 355 360 365 gac aggcaa ggt gag aag aaa ggt gtg ttc tgg tac aac aat aag tta 1152 Asp Arg GlnGly Glu Lys Lys Gly Val Phe Trp Tyr Asn Asn Lys Leu 370 375 380 tga 115514 384 PRT Brassica napus 14 Met Gly Ala Gly Gly Arg Met Gln Val Ser ProPro Ser Lys Lys Ser 1 5 10 15 Glu Thr Asp Thr Ile Lys Arg Val Pro CysGlu Thr Pro Pro Phe Thr 20 25 30 Val Gly Glu Leu Lys Lys Ala Ile Pro ProHis Cys Phe Lys Arg Ser 35 40 45 Ile Pro Arg Ser Phe Ser Tyr Leu Ile TrpAsp Ile Ile Ile Ala Ser 50 55 60 Cys Phe Tyr Tyr Val Ala Thr Thr Tyr PhePro Leu Leu Pro His Pro 65 70 75 80 Leu Ser Tyr Phe Ala Trp Pro Leu TyrTrp Ala Cys Gln Gly Cys Val 85 90 95 Leu Thr Gly Val Trp Val Ile Ala HisGlu Cys Gly His His Ala Phe 100 105 110 Ser Asp Tyr Gln Trp Leu Asp AspThr Val Gly Leu Ile Phe His Ser 115 120 125 Phe Leu Leu Val Pro Tyr PheSer Trp Lys Tyr Ser His Arg Arg His 130 135 140 His Ser Asn Thr Gly SerLeu Glu Arg Asp Glu Val Phe Val Pro Lys 145 150 155 160 Lys Lys Ser AspIle Lys Trp Tyr Gly Lys Tyr Leu Asn Asn Pro Leu 165 170 175 Gly Arg ThrVal Met Leu Thr Val Gln Phe Thr Leu Gly Trp Pro Leu 180 185 190 Tyr LeuAla Phe Asn Val Ser Gly Arg Pro Tyr Asp Gly Gly Phe Ala 195 200 205 CysHis Phe His Pro Asn Ala Pro Ile Tyr Asn Asp Arg Glu Arg Leu 210 215 220Gln Ile Tyr Ile Ser Asp Ala Gly Ile Leu Ala Val Cys Tyr Gly Leu 225 230235 240 Phe Arg Tyr Ala Ala Ala Gln Gly Val Ala Ser Met Val Cys Phe Tyr245 250 255 Gly Val Pro Leu Leu Ile Val Asn Gly Phe Leu Val Leu Ile ThrTyr 260 265 270 Leu Gln His Thr His Pro Ser Leu Pro His Tyr Asp Ser SerGlu Trp 275 280 285 Asp Trp Leu Arg Gly Ala Leu Ala Thr Val Asp Arg AspTyr Gly Ile 290 295 300 Leu Asn Lys Val Phe His Asn Ile Thr Asp Thr HisVal Ala His His 305 310 315 320 Leu Phe Ser Thr Met Pro His Tyr His AlaMet Glu Ala Thr Lys Ala 325 330 335 Ile Lys Pro Ile Leu Gly Glu Tyr TyrGln Phe Asp Gly Thr Pro Val 340 345 350 Val Lys Ala Met Trp Arg Glu AlaLys Glu Cys Ile Tyr Val Glu Pro 355 360 365 Asp Arg Gln Gly Glu Lys LysGly Val Phe Trp Tyr Asn Asn Lys Leu 370 375 380 15 1155 DNA Brassicanapus CDS (1)...(1152) 15 atg ggt gca ggt gga aga atg caa gtg tct cctccc tcc aag aag tct 48 Met Gly Ala Gly Gly Arg Met Gln Val Ser Pro ProSer Lys Lys Ser 1 5 10 15 gaa acc gac acc atc aag cgc gta ccc tgc gagaca ccg ccc ttc act 96 Glu Thr Asp Thr Ile Lys Arg Val Pro Cys Glu ThrPro Pro Phe Thr 20 25 30 gtc gga gaa ctc aag aaa gca atc cca ccg cac tgtttc aaa cgc tcg 144 Val Gly Glu Leu Lys Lys Ala Ile Pro Pro His Cys PheLys Arg Ser 35 40 45 atc cct cgc tct ttc tcc tac ctc atc tgg gac atc atcata gcc tcc 192 Ile Pro Arg Ser Phe Ser Tyr Leu Ile Trp Asp Ile Ile IleAla Ser 50 55 60 tgc ttc tac tac gtc gcc acc act tac ttc cct ctc ctc cctcac cct 240 Cys Phe Tyr Tyr Val Ala Thr Thr Tyr Phe Pro Leu Leu Pro HisPro 65 70 75 80 ctc tcc tac ttc gcc tgg cct ctc tac tgg gcc tgc caa gggtgc gtc 288 Leu Ser Tyr Phe Ala Trp Pro Leu Tyr Trp Ala Cys Gln Gly CysVal 85 90 95 cta acc ggc gtc tgg gtc ata gcc cac gag tgc ggc cac cac gccttc 336 Leu Thr Gly Val Trp Val Ile Ala His Glu Cys Gly His His Ala Phe100 105 110 agc gac tac cag tgg ctt gac gac acc gtc ggt ctc atc ttc cactcc 384 Ser Asp Tyr Gln Trp Leu Asp Asp Thr Val Gly Leu Ile Phe His Ser115 120 125 ttc ctc ctc gtc cct tac ttc tcc tgg aag tac agt cat cga cgccac 432 Phe Leu Leu Val Pro Tyr Phe Ser Trp Lys Tyr Ser His Arg Arg His130 135 140 cat tcc aac act ggc tcc ctc gag aga gac gaa gtg ttt gtc cccaag 480 His Ser Asn Thr Gly Ser Leu Glu Arg Asp Glu Val Phe Val Pro Lys145 150 155 160 aag aag tca gac atc aag tgg tac ggc aag tac cac aac aaccct ttg 528 Lys Lys Ser Asp Ile Lys Trp Tyr Gly Lys Tyr His Asn Asn ProLeu 165 170 175 gga cgc acc gtg atg tta acg gtt cag ttc act ctc ggc tggccg ttg 576 Gly Arg Thr Val Met Leu Thr Val Gln Phe Thr Leu Gly Trp ProLeu 180 185 190 tac tta gcc ttc aac gtc tcg gga aga cct tac gac ggc ggcttc gct 624 Tyr Leu Ala Phe Asn Val Ser Gly Arg Pro Tyr Asp Gly Gly PheAla 195 200 205 tgc cat ttc cac ccc aac gct ccc atc tac aac gac cgc gagcgt ctc 672 Cys His Phe His Pro Asn Ala Pro Ile Tyr Asn Asp Arg Glu ArgLeu 210 215 220 cag ata tac atc tcc gac gct ggc atc ctc gcc gtc tgc tacggt ctc 720 Gln Ile Tyr Ile Ser Asp Ala Gly Ile Leu Ala Val Cys Tyr GlyLeu 225 230 235 240 ttc cgt tac gcc gcc gcg cag gga gtg gcc tcg atg gtctgc ttc tac 768 Phe Arg Tyr Ala Ala Ala Gln Gly Val Ala Ser Met Val CysPhe Tyr 245 250 255 gga gtc ccg ctt ctg att gtc aat ggt ttc ctc gtg ttgatc act tac 816 Gly Val Pro Leu Leu Ile Val Asn Gly Phe Leu Val Leu IleThr Tyr 260 265 270 ttg cag cac acg cat cct tcc ctg cct cac tac gat tcgtcc gag tgg 864 Leu Gln His Thr His Pro Ser Leu Pro His Tyr Asp Ser SerGlu Trp 275 280 285 gat tgg ttg agg gga gct ttg gct acc gtt gac aga gactac gga atc 912 Asp Trp Leu Arg Gly Ala Leu Ala Thr Val Asp Arg Asp TyrGly Ile 290 295 300 ttg aac aag gtc ttc cac aat att acc gac acg cac gtggcg cat cat 960 Leu Asn Lys Val Phe His Asn Ile Thr Asp Thr His Val AlaHis His 305 310 315 320 ctg ttc tcc acg atg ccg cat tat cac gcg atg gaagct acc aag gcg 1008 Leu Phe Ser Thr Met Pro His Tyr His Ala Met Glu AlaThr Lys Ala 325 330 335 ata aag ccg ata ctg gga gag tat tat cag ttc gatggg acg ccg gtg 1056 Ile Lys Pro Ile Leu Gly Glu Tyr Tyr Gln Phe Asp GlyThr Pro Val 340 345 350 gtt aag gcg atg tgg agg gag gcg aag gag tgt atctat gtg gaa ccg 1104 Val Lys Ala Met Trp Arg Glu Ala Lys Glu Cys Ile TyrVal Glu Pro 355 360 365 gac agg caa ggt gag aag aaa ggt gtg ttc tgg tacaac aat aag tta 1152 Asp Arg Gln Gly Glu Lys Lys Gly Val Phe Trp Tyr AsnAsn Lys Leu 370 375 380 tga 1155 16 384 PRT Brassica napus 16 Met GlyAla Gly Gly Arg Met Gln Val Ser Pro Pro Ser Lys Lys Ser 1 5 10 15 GluThr Asp Thr Ile Lys Arg Val Pro Cys Glu Thr Pro Pro Phe Thr 20 25 30 ValGly Glu Leu Lys Lys Ala Ile Pro Pro His Cys Phe Lys Arg Ser 35 40 45 IlePro Arg Ser Phe Ser Tyr Leu Ile Trp Asp Ile Ile Ile Ala Ser 50 55 60 CysPhe Tyr Tyr Val Ala Thr Thr Tyr Phe Pro Leu Leu Pro His Pro 65 70 75 80Leu Ser Tyr Phe Ala Trp Pro Leu Tyr Trp Ala Cys Gln Gly Cys Val 85 90 95Leu Thr Gly Val Trp Val Ile Ala His Glu Cys Gly His His Ala Phe 100 105110 Ser Asp Tyr Gln Trp Leu Asp Asp Thr Val Gly Leu Ile Phe His Ser 115120 125 Phe Leu Leu Val Pro Tyr Phe Ser Trp Lys Tyr Ser His Arg Arg His130 135 140 His Ser Asn Thr Gly Ser Leu Glu Arg Asp Glu Val Phe Val ProLys 145 150 155 160 Lys Lys Ser Asp Ile Lys Trp Tyr Gly Lys Tyr His AsnAsn Pro Leu 165 170 175 Gly Arg Thr Val Met Leu Thr Val Gln Phe Thr LeuGly Trp Pro Leu 180 185 190 Tyr Leu Ala Phe Asn Val Ser Gly Arg Pro TyrAsp Gly Gly Phe Ala 195 200 205 Cys His Phe His Pro Asn Ala Pro Ile TyrAsn Asp Arg Glu Arg Leu 210 215 220 Gln Ile Tyr Ile Ser Asp Ala Gly IleLeu Ala Val Cys Tyr Gly Leu 225 230 235 240 Phe Arg Tyr Ala Ala Ala GlnGly Val Ala Ser Met Val Cys Phe Tyr 245 250 255 Gly Val Pro Leu Leu IleVal Asn Gly Phe Leu Val Leu Ile Thr Tyr 260 265 270 Leu Gln His Thr HisPro Ser Leu Pro His Tyr Asp Ser Ser Glu Trp 275 280 285 Asp Trp Leu ArgGly Ala Leu Ala Thr Val Asp Arg Asp Tyr Gly Ile 290 295 300 Leu Asn LysVal Phe His Asn Ile Thr Asp Thr His Val Ala His His 305 310 315 320 LeuPhe Ser Thr Met Pro His Tyr His Ala Met Glu Ala Thr Lys Ala 325 330 335Ile Lys Pro Ile Leu Gly Glu Tyr Tyr Gln Phe Asp Gly Thr Pro Val 340 345350 Val Lys Ala Met Trp Arg Glu Ala Lys Glu Cys Ile Tyr Val Glu Pro 355360 365 Asp Arg Gln Gly Glu Lys Lys Gly Val Phe Trp Tyr Asn Asn Lys Leu370 375 380 17 1155 DNA Brassica napus CDS (1)...(1152) 17 atg ggt gcaggt gga aga atg caa gtg tct cct ccc tcc aag aag tct 48 Met Gly Ala GlyGly Arg Met Gln Val Ser Pro Pro Ser Lys Lys Ser 1 5 10 15 gaa acc gacacc atc aag cgc gta ccc tgc gag aca ccg ccc ttc act 96 Glu Thr Asp ThrIle Lys Arg Val Pro Cys Glu Thr Pro Pro Phe Thr 20 25 30 gtc gga gaa ctcaag aaa gca atc cca ccg cac tgt ttc aaa cgc tcg 144 Val Gly Glu Leu LysLys Ala Ile Pro Pro His Cys Phe Lys Arg Ser 35 40 45 atc cct cgc tct ttctcc tac ctc atc tgg gac atc atc ata gcc tcc 192 Ile Pro Arg Ser Phe SerTyr Leu Ile Trp Asp Ile Ile Ile Ala Ser 50 55 60 tgc ttc tac tac gtc gccacc act tac ttc cct ctc ctc cct cac cct 240 Cys Phe Tyr Tyr Val Ala ThrThr Tyr Phe Pro Leu Leu Pro His Pro 65 70 75 80 ctc tcc tac ttc gcc tggcct ctc tac tgg gcc tgc caa ggg tgc gtc 288 Leu Ser Tyr Phe Ala Trp ProLeu Tyr Trp Ala Cys Gln Gly Cys Val 85 90 95 cta acc ggc gtc tgg gtc atagcc cac gag tgc ggc cac cac gcc ttc 336 Leu Thr Gly Val Trp Val Ile AlaHis Glu Cys Gly His His Ala Phe 100 105 110 agc gac tac cag tgg ctt gacgac acc gtc ggt ctc atc ttc cac tcc 384 Ser Asp Tyr Gln Trp Leu Asp AspThr Val Gly Leu Ile Phe His Ser 115 120 125 ttc ctc ctc gtc cct tac ttctcc tgg aag tac agt cat cga cgc cac 432 Phe Leu Leu Val Pro Tyr Phe SerTrp Lys Tyr Ser His Arg Arg His 130 135 140 cat tcc aac act ggc tcc ctcgag aga gac gaa gtg ttt gtc ccc aag 480 His Ser Asn Thr Gly Ser Leu GluArg Asp Glu Val Phe Val Pro Lys 145 150 155 160 aag aag tca gac atc aagtgg tac ggc aag tac ctc aac aac cct ttg 528 Lys Lys Ser Asp Ile Lys TrpTyr Gly Lys Tyr Leu Asn Asn Pro Leu 165 170 175 gga cgc acc gtg atg ttaacg gtt cag ttc act ctc ggc tgg ccg ttg 576 Gly Arg Thr Val Met Leu ThrVal Gln Phe Thr Leu Gly Trp Pro Leu 180 185 190 tac tta gcc ttc aac gtctcg gga aga cct tac gac ggc ggc ttc gct 624 Tyr Leu Ala Phe Asn Val SerGly Arg Pro Tyr Asp Gly Gly Phe Ala 195 200 205 tgc cat ttc cac ccc aacgct ccc atc tac aac gac cgc gag cgt ctc 672 Cys His Phe His Pro Asn AlaPro Ile Tyr Asn Asp Arg Glu Arg Leu 210 215 220 cag ata tac atc tcc gacgct ggc atc ctc gcc gtc tgc tac ggt ctc 720 Gln Ile Tyr Ile Ser Asp AlaGly Ile Leu Ala Val Cys Tyr Gly Leu 225 230 235 240 ttc cgt tac gcc gccgcg cag gga gtg gcc tcg atg gtc tgc ttc tac 768 Phe Arg Tyr Ala Ala AlaGln Gly Val Ala Ser Met Val Cys Phe Tyr 245 250 255 gga gtc ccg ctt ctgatt gtc aat ggt ttc ctc gtg ttg atc act tac 816 Gly Val Pro Leu Leu IleVal Asn Gly Phe Leu Val Leu Ile Thr Tyr 260 265 270 ttg cag cac acg catcct tcc ctg cct cac tac gat tcg tcc gag tgg 864 Leu Gln His Thr His ProSer Leu Pro His Tyr Asp Ser Ser Glu Trp 275 280 285 gat tgg ttg agg ggagct ttg gct acc gtt gac aga gac tac gaa atc 912 Asp Trp Leu Arg Gly AlaLeu Ala Thr Val Asp Arg Asp Tyr Glu Ile 290 295 300 ttg aac aag gtc ttccac aat att acc gac acg cac gtg gcg cat cat 960 Leu Asn Lys Val Phe HisAsn Ile Thr Asp Thr His Val Ala His His 305 310 315 320 ctg ttc tcc acgatg ccg cat tat cac gcg atg gaa gct acc aag gcg 1008 Leu Phe Ser Thr MetPro His Tyr His Ala Met Glu Ala Thr Lys Ala 325 330 335 ata aag ccg atactg gga gag tat tat cag ttc gat ggg acg ccg gtg 1056 Ile Lys Pro Ile LeuGly Glu Tyr Tyr Gln Phe Asp Gly Thr Pro Val 340 345 350 gtt aag gcg atgtgg agg gag gcg aag gag tgt atc tat gtg gaa ccg 1104 Val Lys Ala Met TrpArg Glu Ala Lys Glu Cys Ile Tyr Val Glu Pro 355 360 365 gac agg caa ggtgag aag aaa ggt gtg ttc tgg tac aac aat aag tta 1152 Asp Arg Gln Gly GluLys Lys Gly Val Phe Trp Tyr Asn Asn Lys Leu 370 375 380 tga 1155 18 384PRT Brassica napus 18 Met Gly Ala Gly Gly Arg Met Gln Val Ser Pro ProSer Lys Lys Ser 1 5 10 15 Glu Thr Asp Thr Ile Lys Arg Val Pro Cys GluThr Pro Pro Phe Thr 20 25 30 Val Gly Glu Leu Lys Lys Ala Ile Pro Pro HisCys Phe Lys Arg Ser 35 40 45 Ile Pro Arg Ser Phe Ser Tyr Leu Ile Trp AspIle Ile Ile Ala Ser 50 55 60 Cys Phe Tyr Tyr Val Ala Thr Thr Tyr Phe ProLeu Leu Pro His Pro 65 70 75 80 Leu Ser Tyr Phe Ala Trp Pro Leu Tyr TrpAla Cys Gln Gly Cys Val 85 90 95 Leu Thr Gly Val Trp Val Ile Ala His GluCys Gly His His Ala Phe 100 105 110 Ser Asp Tyr Gln Trp Leu Asp Asp ThrVal Gly Leu Ile Phe His Ser 115 120 125 Phe Leu Leu Val Pro Tyr Phe SerTrp Lys Tyr Ser His Arg Arg His 130 135 140 His Ser Asn Thr Gly Ser LeuGlu Arg Asp Glu Val Phe Val Pro Lys 145 150 155 160 Lys Lys Ser Asp IleLys Trp Tyr Gly Lys Tyr Leu Asn Asn Pro Leu 165 170 175 Gly Arg Thr ValMet Leu Thr Val Gln Phe Thr Leu Gly Trp Pro Leu 180 185 190 Tyr Leu AlaPhe Asn Val Ser Gly Arg Pro Tyr Asp Gly Gly Phe Ala 195 200 205 Cys HisPhe His Pro Asn Ala Pro Ile Tyr Asn Asp Arg Glu Arg Leu 210 215 220 GlnIle Tyr Ile Ser Asp Ala Gly Ile Leu Ala Val Cys Tyr Gly Leu 225 230 235240 Phe Arg Tyr Ala Ala Ala Gln Gly Val Ala Ser Met Val Cys Phe Tyr 245250 255 Gly Val Pro Leu Leu Ile Val Asn Gly Phe Leu Val Leu Ile Thr Tyr260 265 270 Leu Gln His Thr His Pro Ser Leu Pro His Tyr Asp Ser Ser GluTrp 275 280 285 Asp Trp Leu Arg Gly Ala Leu Ala Thr Val Asp Arg Asp TyrGlu Ile 290 295 300 Leu Asn Lys Val Phe His Asn Ile Thr Asp Thr His ValAla His His 305 310 315 320 Leu Phe Ser Thr Met Pro His Tyr His Ala MetGlu Ala Thr Lys Ala 325 330 335 Ile Lys Pro Ile Leu Gly Glu Tyr Tyr GlnPhe Asp Gly Thr Pro Val 340 345 350 Val Lys Ala Met Trp Arg Glu Ala LysGlu Cys Ile Tyr Val Glu Pro 355 360 365 Asp Arg Gln Gly Glu Lys Lys GlyVal Phe Trp Tyr Asn Asn Lys Leu 370 375 380 19 21 DNA ArtificialSequence primer 19 ggatatgatg atggtgaaag a 21 20 21 DNA ArtificialSequence primer 20 tctttcacca tcatcatatc c 21 21 21 DNA ArtificialSequence primer 21 gttatgaagc aaagaagaaa c 21 22 26 DNA ArtificialSequence primer 22 gtttcttctt tgctttgctt cataac 26 23 32 DNA ArtificialSequence primer 23 caucaucauc aucttcttcg tagggttcat cg 32 24 33 DNAArtificial Sequence primer 24 cuacuacuac uatcatagaa gagaaaggtt cag 33 2532 DNA Artificial Sequence primer 25 caucaucauc aucatgggtg cacgtggaag aa32 26 33 DNA Artificial Sequence primer 26 cuacuacuac uatctttcaccatcatcata tcc 33 27 30 PRT Arabidopsis thaliana 27 Ile Trp Val Ile AlaHis Glu Cys Gly His His Ala Phe Ser Asp Tyr 1 5 10 15 Gln Trp Leu AspAsp Thr Val Gly Leu Ile Phe His Ser Phe 20 25 30 28 30 PRT Glycine max28 Val Trp Val Ile Ala His Glu Cys Gly His His Ala Phe Ser Lys Tyr 1 510 15 Gln Trp Val Asp Asp Val Val Gly Leu Thr Leu His Ser Thr 20 25 3029 30 PRT Zea mays 29 Val Trp Val Ile Ala His Glu Cys Gly His His AlaPhe Ser Asp Tyr 1 5 10 15 Ser Leu Leu Asp Asp Val Val Gly Leu Val LeuHis Ser Ser 20 25 30 30 29 PRT Ricinus communis 30 Trp Val Met Ala HisAsp Cys Gly His His Ala Phe Ser Asp Tyr Gln 1 5 10 15 Leu Leu Asp AspVal Val Gly Leu Ile Leu His Ser Cys 20 25 31 29 PRT Arabidopsis thaliana31 Leu Leu Val Pro Tyr Phe Ser Trp Lys Tyr Ser His Arg Arg His His 1 510 15 Ser Asn Thr Gly Ser Leu Glu Arg Asp Glu Val Phe Val 20 25 32 29PRT Glycine max 32 Leu Leu Val Pro Tyr Phe Ser Trp Lys Ile Ser His ArgArg His His 1 5 10 15 Ser Asn Thr Gly Ser Leu Asp Arg Asp Glu Val PheVal 20 25 33 29 PRT Zea mays 33 Leu Met Val Pro Tyr Phe Ser Trp Lys TyrSer His Arg Arg His His 1 5 10 15 Ser Asn Thr Gly Ser Leu Glu Arg AspGlu Val Phe Val 20 25 34 29 PRT Ricinus communis 34 Leu Leu Val Pro TyrPhe Ser Trp Lys His Ser His Arg Arg His His 1 5 10 15 Ser Asn Thr GlySer Leu Glu Arg Asp Glu Val Phe Val 20 25 35 36 PRT Arabidopsis thaliana35 Asp Arg Asp Tyr Gly Ile Leu Asn Lys Val Phe His Asn Ile Thr Asp 1 510 15 Thr His Val Ala His His Leu Phe Ser Thr Met Pro His Tyr Asn Ala 2025 30 Met Glu Ala Thr 35 36 36 PRT Glycine max 36 Asp Arg Asp Tyr GlyIle Leu Asn Lys Val Phe His His Ile Thr Asp 1 5 10 15 Thr His Val AlaHis His Leu Phe Ser Thr Met Pro His Tyr His Ala 20 25 30 Met Glu Ala Thr35 37 36 PRT Zea mays 37 Asp Arg Asp Tyr Gly Ile Leu Asn Arg Val Phe HisAsn Ile Thr Asp 1 5 10 15 Thr His Val Ala His His Leu Phe Ser Thr MetPro His Tyr His Ala 20 25 30 Met Glu Ala Thr 35 38 27 PRT Ricinuscommunis 38 Asp Arg Asp Tyr Gly Ile Leu Asn Lys Val Phe His Asn Ile ThrAsp 1 5 10 15 Thr Gln Val Ala His His Leu Phe Thr Met Pro 20 25 39 16PRT Arabidopsis thaliana 39 Ile Lys Trp Tyr Gly Lys Tyr Leu Asn Asn ProLeu Gly Arg Ile Met 1 5 10 15 40 16 PRT Glycine max 40 Val Ala Trp PheSer Leu Tyr Leu Asn Asn Pro Leu Gly Arg Ala Val 1 5 10 15 41 16 PRT Zeamays 41 Pro Trp Tyr Thr Pro Tyr Val Tyr Asn Asn Pro Val Gly Arg Val Val1 5 10 15 42 16 PRT Ricinus communis 42 Ile Arg Trp Tyr Ser Lys Tyr LeuAsn Asn Pro Pro Gly Arg Ile Met 1 5 10 15 43 22 PRT Arabidopsis thaliana43 Trp Ala Leu Phe Val Leu Gly His Asp Cys Gly His Gly Ser Phe Ser 1 510 15 Asn Asp Pro Lys Leu Asn 20 44 22 PRT Brassica napus 44 Trp Ala LeuPhe Val Leu Gly His Asp Cys Gly His Gly Ser Phe Ser 1 5 10 15 Asn AspPro Arg Leu Asn 20 45 22 PRT Glycine max 45 Trp Ala Leu Phe Val Leu GlyHis Asp Cys Gly His Gly Ser Phe Ser 1 5 10 15 Asn Asn Ser Lys Leu Asn 2046 22 PRT Arabidopsis thaliana 46 Trp Ala Ile Phe Val Leu Gly His AspCys Gly His Gly Ser Phe Ser 1 5 10 15 Asp Ile Pro Leu Leu Asn 20 47 10PRT Artificial Sequence exemplary motif 47 Asp Arg Asp Tyr Gly Ile LeuAsn Lys Val 1 5 10 48 22 PRT Glycine max 48 Trp Ala Leu Phe Val Leu GlyHis Asp Cys Gly His Gly Ser Phe Ser 1 5 10 15 Asp Ser Pro Pro Leu Asn 2049 29 PRT Arabidopsis thaliana 49 Ile Leu Val Pro Tyr His Gly Trp ArgIle Ser His Arg Thr His His 1 5 10 15 Gln Asn His Gly His Val Glu AsnAsp Glu Ser Trp His 20 25 50 10 PRT Artificial Sequence exemplary motif50 Asp Arg Asp Tyr Glu Ile Leu Asn Lys Val 1 5 10 51 29 PRT Glycine max51 Ile Leu Val Pro Tyr His Gly Trp Arg Ile Ser His Arg Thr His His 1 510 15 Gln His His Gly His Ala Glu Asn Asp Glu Ser Trp His 20 25 52 29PRT Arabidopsis thaliana 52 Ile Leu Val Pro Tyr His Gly Trp Arg Ile SerHis Arg Thr His His 1 5 10 15 Gln Asn His Gly His Val Glu Asn Asp GluSer Trp Val 20 25 53 6 PRT Artificial Sequence exemplary motif 53 LysTyr His Asn Asn Pro 1 5 54 29 PRT Glycine max 54 Ile Leu Val Pro Tyr HisGly Trp Arg Ile Ser His Arg Thr His His 1 5 10 15 Gln Asn His Gly HisIle Glu Lys Asp Glu Ser Trp Val 20 25 55 6 PRT Brassica napus 55 Gly HisAsp Cys Ala His 1 5 56 6 PRT Brassica napus 56 Gly His Lys Cys Gly His 15 57 6 PRT Brassica napus VARIANT amino acid residues 94-99 ofCanola-Fad3 57 Gly His Asp Cys Gly His 1 5 58 5 PRT Artificial Sequenceexemplary motif 58 His Lys Cys Gly His 1 5 59 6 PRT Artificial Sequenceexemplary motif 59 Ala His Glu Cys Gly His 1 5 60 5 PRT ArtificialSequence exemplary motif 60 His Glu Cys Gly His 1 5 61 5 PRT ArtificialSequence exemplary motif 61 His Arg Arg His His 1 5 62 5 PRT ArtificialSequence exemplary motif 62 His Arg Thr His His 1 5 63 5 PRT ArtificialSequence exemplary motif 63 His Val Ala His His 1 5 64 6 PRT ArtificialSequence exemplary motif 64 Lys Tyr Leu Asn Asn Pro 1 5 65 29 PRTBrassica napus 65 Val Trp Val Ile Ala His Glu Cys Gly His His Ala PheSer Asp Tyr 1 5 10 15 Gln Trp Leu Asp Asp Thr Val Gly Leu Ile Phe HisSer 20 25 66 36 PRT Brassica napus 66 Asp Arg Asp Tyr Gly Ile Leu AsnLys Val Phe His Asn Ile Thr Asp 1 5 10 15 Thr His Val Ala His His LeuPhe Ser Thr Met Pro His Tyr His Ala 20 25 30 Met Glu Ala Thr 35 67 16PRT Brassica napus 67 Ile Lys Trp Tyr Gly Lys Tyr Leu Asn Asn Pro LeuGly Arg Thr Val 1 5 10 15 68 6 PRT Artificial Sequence exemplary motif68 Ala His Lys Cys Gly His 1 5

What is claimed is:
 1. A Brassica plant producing seeds having a longchain monounsaturated fatty acid content of at least about 82% and anerucic acid content of at least about 15% based on total fatty acidcomposition.
 2. The plant of claim 1, said seeds having an oleic acidcontent of at least about 37% based on total fatty acid composition. 3.The plant of claim 1, said seeds having an eicosenoic acid content of atleast about 14% based on total fatty acid composition.
 4. The plant ofclaim 1, wherein said monounsaturated fatty acid content is from about85% to about 90%.
 5. The plant of claim 4, said seeds having an oleicacid content of at least about 42% based on total fatty acidcomposition.
 6. The plant of claim 5, wherein said oleic acid content isfrom about 47% to about 56%.
 7. The plant of claim 4, said seeds havingan erucic acid content of from about 17% to about 31% based on totalfatty acid composition.
 8. The plant of claim 4, said seeds having aneicosenoic acid content from about 15% to about 21% based on total fattyacid composition.
 9. The plant of claim 1, said seeds having a saturatedfatty acid content of less than about 7% based on total fatty acidcomposition.
 10. The plant of claim 1, said seeds having apolyunsaturated fatty acid content of less than about 11% based on totalfatty acid composition.
 11. Progeny of the plant of claim 1, saidprogeny having said long chain monounsaturated fatty acid content andsaid erucic acid content.
 12. A Brassica seed oil having a long chainmonounsaturated fatty acid content of at least about 82% and an erucicacid content of at least about 15% based on total fatty acidcomposition.
 13. The oil of claim 12, said oil having an oleic acidcontent of at least about 37% based on total fatty acid composition. 14.The oil of claim 12, said oil having an eicosenoic acid content of atleast about 14% based on total fatty acid composition.
 15. The oil ofclaim 12, wherein said monounsaturated fatty acid content is from about85% to about 90%.
 16. The oil of claim 15, said oil having an oleic acidcontent of at least about 42% based on total fatty acid composition. 17.The oil of claim 16, wherein said oleic acid content is from about 47%to about 56%.
 18. The oil of claim 15, said oil having an erucic acidcontent of from about 17% to about 31% based on total fatty acidcomposition.
 19. The oil of claim 15, said oil having an eicosenoic acidcontent from about 15% to about 21% based on total fatty acidcomposition.
 20. The oil of claim 12, said oil having a saturated fattyacid content of less than about 7% based on total fatty acidcomposition.
 21. The oil of claim 12, said oil having a polyunsaturatedfatty acid content of less than about 11% based on total fatty acidcomposition.
 22. The oil of claim 21, wherein said polyunsaturated fattyacid content is less than about 9%.
 23. A method of producing a planthaving a long chain monounsaturated fatty acid content of at least about82% and an erucic acid content of at least about 15% based on totalfatty acid composition, said method comprising the steps of crossing afirst plant line with a second plant line and selecting progeny of saidcross having said monounsaturated content, wherein said first plant linehas an erucic acid content of at least about 45% based on total fattyacid composition and said second plant line has an oleic acid content ofat least about 84% based on total fatty acid composition.